. 2023 Sep 13;21(9):e08215. doi: 10.2903/j.efsa.2023.8215

Table A.3.

Repeated dose toxicity studies

Test substance	Exposure conditions	Result	Reference
White P7H MO Paraffinic, low viscosityn‐C14 to n‐C32 (average C20) 48% was </= C20	F344 rats (f), age: 4 weeks, 10 per group Dose/route: 0, 0.02, 0.2 or 2% in feed, corresponding to 0, 17, 170 or 1,690 mg/kg bw per day (reported in the publication) Duration: 90 days	NOAEL 17 mg/kg bw per day Tissue levels at 0, 17, 170, 1,690 mg/kg bw per day: Liver: ND, ND, 2,280 and 3,000 mg/kg, MLN: ND, ND, 2,910 and 1,860 mg/kg, Spleen: ND at all doses, Effects: Liver: abs weight ↑ ^(a) (mid and top dose), incidence of granulomas ↑ (top dose) Immune system: abs number of neutrophiles ↑ (top dose) MLN: weight ↑ and inflammation (mid and top dose), incidence ↑ and severity of granulomas (mid and top dose) Spleen: weight ↔, histopathology unchanged	McKee et al. (2012)
Experiment broad MOSH mixture: Broad white MO mixture n‐C14 – n‐C50	F344 rats (f), age: 4 weeks, 25 per group (5 rats/time point) Dose/route: 0, 400, 1,000 or 4,000 mg/kg in feed, corresponding to 0, 2.6, 25 or 260 mg/kg bw per day (calculated on the basis of individual body weights and cage averaged feed intake) Background contamination of control feed: 1.6 mg/kg feed Duration: 30, 60, 90, 120 days or 120 + 30 days of control feed for depuration	NOAEL 25 mg/kg bw per day Tissue levels at exposure to 0, 2.6., 25, 260 mg/kg bw per day after 90 days: Liver: 30, 238, 1,411 and 5,492 mg/kg MLN: not analysed Spleen: 8, 31, 128 and 274 mg/kg Effects: Liver: abs weight ↑ (top dose), density of granulomas ↑ (top dose) Immune system: lymphoid cells in liver parenchyma ↑ (top dose) MLN: not analysed Spleen: weight ↔ Clinical chemistry and haematology analyses not performed	Nygaard et al. (2023), Barp et al. (2017a, 2017b)
Experiment 3 narrow MOSH mixtures ○ S‐C25: n‐C16 – n‐C34, 73% ≤ n‐C25, incl. paraffins ○ L‐C25: n‐C25 to n‐C50, deparaffinated ○ L‐C25W: 1:1 (w:w) mixture L‐C25 and wax: 80% n‐C23 to n‐C45, incl paraffins	F344 rats (f), age: 4 weeks, 8 per group Dose/route: 0, 400, 1,000 or 4,000 mg/kg in feed, corresponding to S‐C25: 0, 23, 61 or 226 mg/kg bw per day L‐C25: 0, 25, 62 or 236 mg/kg bw per day L‐C25W: 0, 22, 53 or 218 mg/kg bw per day (calculated on the basis of individual body weights and cage averaged feed intake) Background contamination of control feed: 1.5 mg/kg feed Duration: 120 days	S‐C25, LOAEL 23 mg/kg bw per day L‐C25, NOAEL 236 mg/kg bw per day LC25W, LOAEL 22 mg/kg bw per day Tissue levels after 90d exposure: Liver: S‐C25: 16.5, 4,784, 9,223 and 14,639 mg/kg at 0, 20, 52 and 222 mg/kg bw per day, respectively L‐C25: 16.5, 1,393, 2,590 and 3,805 mg/kg at 0, 22, 54 and 207 mg/kg bw per day, respectively L‐C25W: 16.5, 4,827, 6,851 and 7,709 mg/kg at 0, 19, 46 and 187 mg/kg bw per day, respectively MLN: Not analysed Spleen: S‐C25: 1.3, 287, 538 and 499 mg/kg at 0, 20, 52 and 222 mg/kg bw per day, respectively L‐C25: 1.3, 192, 263 and 419 mg/kg at 0, 22, 54 and 207 mg/kg bw per day, respectively L‐C25W: 1.3, 134, 147 and 243 mg/kg at 0, 19, 46 and 187 mg/kg bw per day, respectively Effects: Liver: S‐C25: relative weight ↑ (all doses), absolute weight ↑ (top dose), density of granulomas ↑ (top dose) L‐C25: No findings L‐C25W: absolute weight ↑ (all doses), density of granulomas ↑ (all doses) MLN: Not analysed Immune system: S‐C25: Lymphoid cells in liver parenchyma ↑ (top dose) L‐C25: No findings L‐C25W: ↑ (all doses) Spleen: S‐C25: absolute weight ↑ (all doses) L‐C25: No findings L‐C25W: absolute weight ↑ (all doses) Clinical chemistry and haematology analyses not performed
GTL base oil experiment: GTL base oil (n‐C18 to n‐C50, branched and linear alkanes) dissolved in arachis oil	OECD testing guideline 408 SD rats (m + f), age: unknown, 20 per group Dose/route: 0, 50, 200 or 1,000 mg/kg bw per day by gavage Duration: 90 days, 90 at highest dose+28 days of control feed for depuration	Effects: No toxicity observed, some accumulation of alveolar macrophages with vacuolated cytoplasm in lungs (two top doses) Female rats: MLN showed vacuolated histiocytes (both effects not considered biologically relevant by the study authors)	Boogaard et al. (2017)
Follow up 28‐day study: GTL base oil n‐C18 to n‐C30	Rats (gender unknown), age: unknown, number per group: unknown Dose/route: 0, 750, 3,750, 15,000 mg/kg feed, corresponding to 0, 63, 308 and 1,267 mg/kg bw per day (reported in the publication) Duration: 28 days	Effects: Lungs: No effects mild to moderate apoptosis and necrosis in lamina propria, crypts of duodenum, jejunum and ileum in male rats (top dose) Spleen: lymphocytosis in males (minimal in top dose) MLN and Peyer's patches: lymphocytosis in males (minimal in low and mid dose, moderate at top dose)
GTL residual oil experiment: GTL residual oil (n‐C40 to nC70, branched, cyclic and linear alkanes) dissolved in polyethylene glycol 400	OECD testing Guideline 407 Wistar rats (m + f), age: unknown, 10 per group Dose/route: 0, 30, 300, 1,000 mg/kg bw per day by gavage Duration: 28 days, 28 at highest dose +14 days of control feed for depuration	NOAEL 1,000 mg/kg bw Effects: Haematopoiesis in spleen of female rats (two top doses), effect reversible. Considered adaptive, not adverse.
90‐d dietary repeated‐dose toxicity study: GTL waxes (n‐C15 to n‐C50, branched and linear alkanes) Sarawaxes – SX30 (C18–C25) – SX50 (C19–C36) – SX701 (C25–C48)	F344 rats (f), age: unknown, 5–10 per group Doses/route: SX30, SX50, SX701: 0, 0.002%, 0.02%, 0.2%, 2% in feed, corresponding to 0, 1.8, 18, 180, 1,800 mg/kg bw per day SX701: 5% in feed, corresponding to 4,500 mg/kg bw per day (calculated by EFSA default values) Duration: 90 days SD rats (unknown), age: unknown, 5 per group Dose/route: SX30, SX50, SX701: 0%, 2% in feed, corresponding to 0, 1,800 mg/kg bw per day (calculated by EFSA default values) Duration: 90 days	Effects: Liver: absolute and relative liver weight ↑ (F344 at SX50 2% and at SX701 2% and 5%), relative liver weight ↑ (SD at SX30 2%) MLN: absolute and relative weight ↑ (F344 at SX30 2%, SX50 0.2 and 2%, SX701 0.02, 0.2, 2 and 5%, SD at SX30 and SX50 doses unknown), absolute weight ↑ (SD at SX701) Spleen: ↑ (F344 at SX30 2%, SX50 0.2% and 2%, SX701 0.2%, 2%, 5%), absolute and relative weight ↔ (SD rats) Microgranulomata in liver: present (F344 at SX50 and SX701 > 0.2%) Periportal vacuolisation: present (F344 at SX701 2% and 5%) Foci of inflammatory cells: present (F344 at SX701 0.002% and 0.02%, SD at SX50 and SX701 2%) Liver lesions: None (F344 and SD rats at SX30) MLN lesions, reactive nodes, histiocytosis, adenitis: present (F344 at SX30, SX50, SX701 at 2%, SX701 at 5%), histiocytosis (F344 at SX50, SX701 lower doses) Heart lesions, thickening, chronic inflammation, ↑ basophilia of mitral valve: (F344 SX50 2%, SX701 2% and 5%) Accumulation of non‐polar hydrocarbon material in liver: (F344 rats at SX50 and SX701), in MLN: (F344 at SX30, SX50, SX701)
90‐d repeated‐dose study: GTL waxes (n‐C15 to n‐C50, branched and linear alkanes) Sarawaxes – SX701 (C25‐C48 – SX701 (C25‐C48) – SX100 (C38‐C90)	SD and F334 rats (f), age: unknown, 5–10 per group Doses/route: SX701: 0, 2, 5% in feed, corresponding to 0, 1,800, 4,500 mg/kg bw per day SX702, SX100: 0, 0.002, 0.02, 0.2, 2% in feed, corresponding to 0, 1.8, 18, 180, 1,800 mg/kg bw per day (calculated by EFSA default values) Duration: 90 days F344 (f), age: unknown, 5 per group Doses/route: SX701: 0, 5% in feed, corresponding to 0, 4,500 mg/kg bw per day (calculated by EFSA default values) Duration: 4, 8, 13 week, 13 week + 6–12 weeks reversal SD rats (f), age: unknown, 5 per group Doses/route: SX701: 0, 2% in feed, corresponding to 0, 1,800 mg/kg bw per day (calculated by EFSA default values) Duration: 13 week	Effects: Appearance: Red staining in face, genital area, unkempt fur (F344 at SX701 5%, 13 w, improved in reversal period) Liver and spleen: Absolute and relative weight ↑ (F344), ↔ (SD) MLN: absolute and relative weight ↑ (F344, SD) Spleen: Absolute and relative weight ↑ (F344), ↔ (SD) Lesions in liver, MLN, mitral valve of heart: (F344 at SX701) Lesions in MLN: (F344 at SX702 0.02, 0.2, 2%) Severity all lesions: ↓ in reversal period SX100 no evidence of lesions
90‐day repeated dose study: Paraffin wax (FCM 93 58)	SD rats (m + f), age 53–55 days old at the start of administration, 10 animals/sex per group. Doses/route Oral via the diet Four treatment groups: 0, 0.2, 2 or 10 g/kg bw per day (nominal doses), plus two groups (0 and 10 g/kg bw per day) allowed for 6‐week recovery after the 13‐week administration period. Measured doses: m: 0.19, 1.9, and 9 g/kg bw per day; f: 0.24, 2.0 and 10 g/kg bw per day. Duration: 13 weeks +6 weeks of recovery (only control and top dose groups) Study parameters compliant with OECD 408 (2018)	NOAEL: 9 g/kg bw per day Effects: No adverse effects observed up to the highest tested dose. Increased MLN weights observed in both sexes (achieving statistical significance in female rats at all tested doses). Macrophage aggregates were observed in MLN of females rats with incidence and severity increasing with dosage.	ERBC Study A4471 (2022)

Test substance

Exposure conditions

Result

Reference

White P7H MO

Paraffinic, low viscosityn‐C14 to n‐C32 (average C20)

48% was </= C20

F344 rats (f), age: 4 weeks, 10 per group

Dose/route: 0, 0.02, 0.2 or 2% in feed, corresponding to 0, 17, 170 or 1,690 mg/kg bw per day (reported in the publication)

Duration: 90 days

NOAEL 17 mg/kg bw per day

Tissue levels at 0, 17, 170, 1,690 mg/kg bw per day:

Liver: ND, ND, 2,280 and 3,000 mg/kg,

MLN: ND, ND, 2,910 and 1,860 mg/kg,

Spleen: ND at all doses,

Effects: Liver: abs weight ↑ ^(a) (mid and top dose), incidence of granulomas ↑ (top dose)

Immune system: abs number of neutrophiles ↑ (top dose)

MLN: weight ↑ and inflammation (mid and top dose), incidence ↑ and severity of granulomas (mid and top dose)

Spleen: weight ↔, histopathology unchanged

McKee et al. (2012)

Experiment broad MOSH mixture:

Broad white MO mixture

n‐C14 – n‐C50

F344 rats (f), age: 4 weeks, 25 per group (5 rats/time point)

Dose/route: 0, 400, 1,000 or 4,000 mg/kg in feed, corresponding to 0, 2.6, 25 or 260 mg/kg bw per day (calculated on the basis of individual body weights and cage averaged feed intake)

Background contamination of control feed: 1.6 mg/kg feed

Duration: 30, 60, 90, 120 days or 120 + 30 days of control feed for depuration

NOAEL 25 mg/kg bw per day

Tissue levels at exposure to 0, 2.6., 25, 260 mg/kg bw per day after 90 days: Liver: 30, 238, 1,411 and 5,492 mg/kg MLN: not analysed

Spleen: 8, 31, 128 and 274 mg/kg

Effects:

Liver: abs weight ↑ (top dose), density of granulomas ↑ (top dose)

Immune system: lymphoid cells in liver parenchyma ↑ (top dose)

MLN: not analysed

Spleen: weight ↔

Clinical chemistry and haematology analyses not performed

Nygaard et al. (2023),

Barp et al. (2017a, 2017b)

Experiment 3 narrow MOSH mixtures

○
S‐C25: n‐C16 – n‐C34, 73% ≤ n‐C25, incl. paraffins
○
L‐C25: n‐C25 to n‐C50, deparaffinated
○
L‐C25W: 1:1 (w:w) mixture L‐C25 and wax: 80% n‐C23 to n‐C45, incl paraffins

F344 rats (f), age: 4 weeks, 8 per group

Dose/route: 0, 400, 1,000 or 4,000 mg/kg in feed, corresponding to

S‐C25: 0, 23, 61 or 226 mg/kg bw per day

L‐C25: 0, 25, 62 or 236 mg/kg bw per day

L‐C25W: 0, 22, 53 or 218 mg/kg bw per day (calculated on the basis of individual body weights and cage averaged feed intake)

Background contamination of control feed: 1.5 mg/kg feed

Duration: 120 days

S‐C25, LOAEL 23 mg/kg bw per day

L‐C25, NOAEL 236 mg/kg bw per day

LC25W, LOAEL 22 mg/kg bw per day

Tissue levels after 90d exposure:

Liver:

S‐C25: 16.5, 4,784, 9,223 and 14,639 mg/kg at 0, 20, 52 and 222 mg/kg bw per day, respectively

L‐C25: 16.5, 1,393, 2,590 and 3,805 mg/kg at 0, 22, 54 and 207 mg/kg bw per day, respectively

L‐C25W: 16.5, 4,827, 6,851 and 7,709 mg/kg at 0, 19, 46 and 187 mg/kg bw per day, respectively

MLN: Not analysed

Spleen:

S‐C25: 1.3, 287, 538 and 499 mg/kg at 0, 20, 52 and 222 mg/kg bw per day, respectively

L‐C25: 1.3, 192, 263 and 419 mg/kg at 0, 22, 54 and 207 mg/kg bw per day, respectively

L‐C25W: 1.3, 134, 147 and 243 mg/kg at 0, 19, 46 and 187 mg/kg bw per day, respectively

Effects:

Liver:

S‐C25: relative weight ↑ (all doses), absolute weight ↑ (top dose), density of granulomas ↑ (top dose)

L‐C25: No findings

L‐C25W: absolute weight ↑ (all doses), density of granulomas ↑ (all doses)

MLN: Not analysed

Immune system:

S‐C25: Lymphoid cells in liver parenchyma ↑ (top dose)

L‐C25: No findings

L‐C25W: ↑ (all doses)

Spleen:

S‐C25: absolute weight ↑ (all doses)

L‐C25: No findings

L‐C25W: absolute weight ↑ (all doses)

Clinical chemistry and haematology analyses not performed

GTL base oil experiment: GTL base oil (n‐C18 to n‐C50, branched and linear alkanes) dissolved in arachis oil

OECD testing guideline 408

SD rats (m + f), age: unknown, 20 per group

Dose/route: 0, 50, 200 or 1,000 mg/kg bw per day by gavage

Duration: 90 days, 90 at highest dose+28 days of control feed for depuration

Effects: No toxicity observed, some accumulation of alveolar macrophages with vacuolated cytoplasm in lungs (two top doses)

Female rats: MLN showed vacuolated histiocytes

(both effects not considered biologically relevant by the study authors)

Boogaard et al. (2017)

Follow up 28‐day study: GTL base oil n‐C18 to n‐C30

Rats (gender unknown), age: unknown, number per group: unknown

Dose/route: 0, 750, 3,750, 15,000 mg/kg feed, corresponding to 0, 63, 308 and 1,267 mg/kg bw per day (reported in the publication)

Duration: 28 days

Effects: Lungs: No effects mild to moderate apoptosis and necrosis in lamina propria, crypts of duodenum, jejunum and ileum in male rats (top dose)

Spleen: lymphocytosis in males (minimal in top dose)

MLN and Peyer's patches: lymphocytosis in males (minimal in low and mid dose, moderate at top dose)

GTL residual oil experiment:

GTL residual oil (n‐C40 to nC70, branched, cyclic and linear alkanes) dissolved in polyethylene glycol 400

OECD testing Guideline 407

Wistar rats (m + f), age: unknown, 10 per group

Dose/route: 0, 30, 300, 1,000 mg/kg bw per day by gavage

Duration: 28 days, 28 at highest dose +14 days of control feed for depuration

NOAEL 1,000 mg/kg bw

Effects: Haematopoiesis in spleen of female rats (two top doses), effect reversible.

Considered adaptive, not adverse.

90‐d dietary repeated‐dose toxicity study:

GTL waxes (n‐C15 to n‐C50, branched and linear alkanes)

Sarawaxes

–
SX30 (C18–C25)
–
SX50 (C19–C36)
–
SX701 (C25–C48)

F344 rats (f), age: unknown, 5–10 per group

Doses/route: SX30, SX50, SX701:

0, 0.002%, 0.02%, 0.2%, 2% in feed, corresponding to 0, 1.8, 18, 180, 1,800 mg/kg bw per day

SX701: 5% in feed, corresponding to 4,500 mg/kg bw per day (calculated by EFSA default values)

Duration: 90 days

SD rats (unknown), age: unknown, 5 per group

Dose/route: SX30, SX50, SX701: 0%, 2% in feed, corresponding to 0, 1,800 mg/kg bw per day (calculated by EFSA default values)

Duration: 90 days

Effects: Liver: absolute and relative liver weight ↑ (F344 at SX50 2% and at SX701 2% and 5%), relative liver weight ↑ (SD at SX30 2%)

MLN: absolute and relative weight ↑ (F344 at SX30 2%, SX50 0.2 and 2%, SX701 0.02, 0.2, 2 and 5%, SD at SX30 and SX50 doses unknown), absolute weight ↑ (SD at SX701)

Spleen: ↑ (F344 at SX30 2%, SX50 0.2% and 2%, SX701 0.2%, 2%, 5%), absolute and relative weight ↔ (SD rats)

Microgranulomata in liver: present (F344 at SX50 and SX701 > 0.2%)

Periportal vacuolisation: present (F344 at SX701 2% and 5%)

Foci of inflammatory cells: present (F344 at SX701 0.002% and 0.02%, SD at SX50 and SX701 2%)

Liver lesions: None (F344 and SD rats at SX30)

MLN lesions, reactive nodes, histiocytosis, adenitis: present (F344 at SX30, SX50, SX701 at 2%, SX701 at 5%), histiocytosis (F344 at SX50, SX701 lower doses)

Heart lesions, thickening, chronic inflammation, ↑ basophilia of mitral valve: (F344 SX50 2%, SX701 2% and 5%)

Accumulation of non‐polar hydrocarbon material in liver: (F344 rats at SX50 and SX701), in MLN: (F344 at SX30, SX50, SX701)

90‐d repeated‐dose study:

GTL waxes (n‐C15 to n‐C50, branched and linear alkanes)

Sarawaxes

–
SX701 (C25‐C48
–
SX701 (C25‐C48)
–
SX100 (C38‐C90)

SD and F334 rats (f), age: unknown, 5–10 per group

Doses/route:

SX701: 0, 2, 5% in feed, corresponding to 0, 1,800, 4,500 mg/kg bw per day

SX702, SX100: 0, 0.002, 0.02, 0.2, 2% in feed, corresponding to 0, 1.8, 18, 180, 1,800 mg/kg bw per day (calculated by EFSA default values)

Duration: 90 days

F344 (f), age: unknown, 5 per group

Doses/route:

SX701: 0, 5% in feed, corresponding to 0, 4,500 mg/kg bw per day (calculated by EFSA default values)

Duration: 4, 8, 13 week, 13 week + 6–12 weeks reversal

SD rats (f), age: unknown, 5 per group

Doses/route:

SX701: 0, 2% in feed, corresponding to 0, 1,800 mg/kg bw per day (calculated by EFSA default values)

Duration: 13 week

Effects: Appearance: Red staining in face, genital area, unkempt fur (F344 at SX701 5%, 13 w, improved in reversal period)

Liver and spleen: Absolute and relative weight ↑ (F344), ↔ (SD)

MLN: absolute and relative weight ↑ (F344, SD)

Spleen: Absolute and relative weight ↑ (F344), ↔ (SD)

Lesions in liver, MLN, mitral valve of heart: (F344 at SX701)

Lesions in MLN: (F344 at SX702 0.02, 0.2, 2%)

Severity all lesions: ↓ in reversal period

SX100 no evidence of lesions

90‐day repeated dose study:

Paraffin wax (FCM 93 58)

SD rats (m + f), age 53–55 days old at the start of administration, 10 animals/sex per group.

Doses/route

Oral via the diet

Four treatment groups: 0, 0.2, 2 or 10 g/kg bw per day (nominal doses), plus two groups (0 and 10 g/kg bw per day) allowed for 6‐week recovery after the 13‐week administration period.

Measured doses:

m: 0.19, 1.9, and 9 g/kg bw per day;

f: 0.24, 2.0 and 10 g/kg bw per day.

Duration:

13 weeks +6 weeks of recovery (only control and top dose groups)

Study parameters compliant with OECD 408 (2018)

NOAEL: 9 g/kg bw per day

Effects: No adverse effects observed up to the highest tested dose. Increased MLN weights observed in both sexes (achieving statistical significance in female rats at all tested doses). Macrophage aggregates were observed in MLN of females rats with incidence and severity increasing with dosage.

ERBC Study A4471 (2022)

^(a)

↑ indicates an increase, ↓ a decrease, ↔ indicates no changes.