4,6-bis(octylthiomethyl)-o-cresol

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

yes

Remarks:

The study was set up as a two-generation-study (OECD 416). In the absence of findings, it was discontinued on day 21 of the F1 generation.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,6-bis(octylthiomethyl)-o-cresol

EC Number:

402-860-6

EC Name:

4,6-bis(octylthiomethyl)-o-cresol

Cas Number:

110553-27-0

Molecular formula:

C25 H44 O S2

IUPAC Name:

2-methyl-4,6-bis[(octylsulfanyl)methyl]phenol

Details on test material:

- Storage condition of test material: at room temperature in the dark

Test animals

Species:

rat

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: supplied by Harlan Nossan S.r.l., San Pietro al Natisone (UD) Italy
- Age at study initiation: (P) 6-7 weeks and 8 to 9 weeks for males and females, respectively
- Weight at study initiation: the animals used; a total of 110 male and 110 female rats, were within a 25 gram weight range (P)
- Housing: (a) during the pre-mating period in clear polycarbonate cages measuring 59x39x20 cm with a stainless steel mesh lid and floor (Type 4: Techniplast Gazzada S.r.l., Buguggiate, Varese; each cage tray held absorbent material which was inspected daily and changed at least 3 times per week); (b) during the mating period on the basis of one male to one female in clear polycarbonate cages measuring 43x27x15 cm with a stainless steel mesh lid and floor (Type 3: Techniplast Gazzada S.r.l; each cage tray held absorbent material which was inspected and changed daily. The males were re-caged after mating as they were before mating. The mated females were caged in individual, solid bottomed, breeding cages (Type 3: Techniplast Gazzada S.r.l.).
- Diet (ad libitum): commercially available rodent diet (Altromin MT, Altromin-Rieper, Bolzano, Italy)
- Water (ad libitum): potable water
- Acclimation period: at leat 11 days; the male animals arrived on 8 October 1999 and were allocated to groups on 15 October 1999. The females arrived on 3 December 1999 and were allocated to groups on 10 December 1999. The first day of treatment for males was on 21 October 1999. The first day of treatment for females was on 16 December 1999 (the last sacrifice was performed on 3 April 2000).

ENVIRONMENTAL CONDITIONS
The animals were housed in the barriered rodent facility at RTC. Animal room controls were set
- Temperature (°C): 22±3
- Humidity (%): 30±70
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

peanut oil

Remarks:

a weighed amount of the test substance was suspended in the vehicle and brought to the final volume appropriate for each concentration

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test suspensions were prepared daily at room temperature
- Males: dose volumes were calculated according to individual body weights on the first day of treatment and adjusted according to individual body weights at weekly intervals thereafter.
- Females: dose volumes were calculated according to individual body weights at weekly intervals before pairing and on Days 0, 6, 10 and 15 post-coitum. Thereafter individual dose volumes remained constant

VEHICLE
- Concentration in vehicle: 0, 3, 12.5 or 40 mg/ml, respectively for the 0, 15, 50 and 200 mg/kg bw dose groups
- Amount of vehicle (if gavage): 5 ml/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 4 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to commencement of treatment the proposed formulation procedure was checked by chemical analysis to confirm that the method was acceptable. Samples of formulations taken during the first and last week of treatment were also analysed for concentration homogeneity and stability. All analytical results were in the acceptable range.

Duration of treatment / exposure:

- Males: a total of approx. 113 days (for 10 weeks prior to pairing, through the mating period and thereafter until the day prior to sacrifice)
- Females: a total of approx. 56 days (for 2 weeks prior to pairing, during the mating period and through to weaning of the offspring)

Frequency of treatment:

Daily

Details on study schedule:

- Selection of parents from F1 generation: since no signs of toxicity were observed, the Sponsor requested that the study be stopped at the first generation after weaning and selection of the F1 generation.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: chosen because of the results of 28-day and 90-day toxicity studies, as well as the teratogenicity study performed in rats. In the 28-day oral toxicity study (gavage) the NOEL was 50 mg/kg body weight, with increased alkaline phosphatase and liver weight increase in the 250 and 1000 mg/kg bw dose groups. In the 90-day oral toxicity study in rats (gavage) the NOEL was 10 mg/kg bw, with increased liver weights at 100 and 1000 mg/kg bw. In the rat teratology study 150 mg/kg bw (gavage) represented the overall NOEL, with reduced body weight gain at 300 mg/kg bw of the pregnant dams.
Therefore, since animals were dosed for at least 100 days, 200 mg/kg was considered to yield a maximum tolerated dose, while 15 mg/kg was expected to yield a NOEL with 50 mg/kg to show intermediate toxicity.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS, DETAILED CLINICAL OBSERVATIONS: Yes
- Mortality: all cages were checked for dead or moribund animals twice daily throughout the study, once in the morning and again in the afternoon. A similar procedure was followed at weekends and Public Holidays except that the female check was carried out at approximately mid-day.
- Clinical signs: all signs were recorded for individual animals. During the treatment period, for both F0 males and females, examination of individual animals for signs of reaction to treatment was carried out daily prior to dosing, immediately after and at approximately 1 hour after dosing. All animals received a more thorough examination once weekly during the entire treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: males were weighed on the day of allocation, the day treatment commenced, and weekly thereafter and just prior to sacrifice. Females were weighed on the day of allocation, the day treatment commenced and weekly to pairing. During the post-coitum period females were weighed on Days 0, 6, 10, 15, and 20 post-coitum and also on Days 0/1, 4, 8, 12 and 21 post-partum.

FOOD CONSUMPTION:
Food consumption per cage of animals was measured weekly for males from allocation to pairing and for females from the day of allocation to pairing. Individual food consumption of females was measured over the periods of Days 0 to 5, 6 to 9, 10 to 14 and 15 to 19 post-coitum, and Days 0 to 3, 4 to 7, 8 to 11 and 12 to 20 postpartum.

Oestrous cyclicity (parental animals):

- Oestrous cycles and mating performance: vaginal smears were taken daily for 2 weeks prior to pairing starting from the first day of treatment until occurrence of mating. During the mating period each cage was checked each morning for the presence of a copulation plug and a vaginal smear was prepared from each female. This information was used to detect marked anomalies of the oestrus cycle and to determine the precoital interval (the number of nights paired before detection of mating).
-Duration of gestation: gestation periods were taken as the time between the day of successful mating and the commencement of birth (i.e. first detected presence of offspring).

Sperm parameters (parental animals):

(Only F0 males) Sperm count in one epididymis and evaluation of sperm viability and motility was performed in control and high-dose males and in one mid-dose male which did not induce pregnancy

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- On Day 4 post-partum, all litters in excess of eight offspring were culled to eight pups (4 males and 4 females, where possible) by random selection, excluding those pups showing signs of ill health. Culled pups were sacrificed on Day 4 post-partum by subcutaneous injection of 'Tanax', necropsied and examined for external and internal abnormalities. Pup sex was confirmed at necropsy. One litter was inadvertently culled to seven pups.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
The pups (live and dead) were counted, sexed, weighed and examined for external abnormalities as soon as possible after parturition (Day 0 or 1 post-partum). Live pups were identified individually within the litter by toe amputation. All litters were examined daily for dead or abnormal young. The pups were also weighed on Days 4, 8, 12 and 21 post-partum. All pups found dead were given a post-mortem examination.
- 24 males and 24 females were selected for further study. All pups were allocated to 4 groups and treated starting the day after weaning. Clinical observation, post dose observation, food consumption, body weight and post weaning tests were performed. Meanwhile the Sponsor decided to stop the study at the first generation, since no toxicity was observed.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

ASSESSMENT OF PRE-WEANING DEVELOPMENT
All pups in all litters were examined to determine the age at which the following developmental stages were attained:
a) Pinna unfolding, from Day 1 post-partum to 100% occurrence;
b) Hair growth, from Day 3 post-partum to 100% occurrence;
c) Incisor emption (upper), from Day 8 post-partum to 100% occurrence;
d) Startle response to sound, from Day 12 post-partum to 100% occurrence;
e) Eye opening (complete separation of eyelids), from Day 12 post-partum to 100% occurrence;
f) Air righting reflex, once on Day 20 post-partum;
g) Pupil reflex, once on Day 20 post-partum;
h) Testes descent (testes palpable in the scrotum), once on Day 21 post-partum.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: males were killed by carbon dioxide after the birth of the majority of litters and retained for external and internal examinations.
- Maternal animals: all parental animals were killed at weaning (PND 21) by carbon dioxide inhalation. Apparently non pregnant females were sacrificed after at least 25 days post-coitum.

GROSS NECROPSY
- Gross necropsy consisted of and examined for external and internal abnormalities
- The sex of each pup was determined by gonadal inspection and, for each dam, the number of visible implantation sites was recorded

HISTOPATHOLOGY / ORGAN WEIGHTS
- Females: uteri were immersed in a 10% solution of ammonium sulphide to reveal any evidence of implantations. The following tissues were preserved in 10% buffered formol saline: uterus with cervix, ovaries with oviduct, vagina and pituitary
- Males: the following tissues were preserved in 10% buffered formol saline (except testes and one epididymis which were fixed in Bouin's fluid and preserved in 70% alcohol): pituitary, testes, one epididymis, seminal vesicles, prostate, coagulating gland and any Abnormalities. Testes from males which did not induce pregnancy and from 5 concurrent controls were weighed.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 4 days of age.
- All pups found dead in the cage were necropsied.
- Excess pups (i.e., those not selected for the Fl generation) were killed with carbon dioxide on or shortly after Day 21 post-partum
- All animals were killed with carbon dioxide and were subjected to necropsy

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- Culled pups were sacrificed by intrascapular injection of Tanax on the day of culling and were subjected to a post-mortem examination. The sex of each pup was determined by gonadal inspection.

Statistics:

For the body weights, body weight gain and organ weights the significance of the differences amongst group means was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett's test using a pooled error of variance. The homogeneity of the data were assessed by Bartlett's Test before Dunnett's Test was performed. If the data were found to be inhomogeneous, a modified t test (Cochran and Cox) was applied.
The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
The non-parametric Kruskal-Wallis analysis of variance was used for litter data.
Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams' test.
The criterion for statistical significance was p<0.05.

Reproductive indices:

mating index, copulation index, fertilitiy index

Offspring viability indices:

viability index, lactation index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs seen during the observations performed at weekly intervals in F0 males and females were limited to common conditions of the skin and fur. No reaction to treatment was seen at the post-dose observations performed immediately and 1 hour after dosing during the entire treatment period except for a few occasions of salivation in the high-dose group.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control male was found dead and another one was sacrificed for humane reasons during the course of the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Group mean body weights and body weight gain in F0 males were comparable between the treated and the control groups. Statistically significant reductions in body weight were observed on day 8 of treatment in females receiving 15 and 50 mg/kg/day of the test substance when compared to controls. A statistically significant reduction in body weight gain was observed in females receiving the test substance at 50 mg/kg/day on day 8 of treatment. Body weight gain in females receiving the test substance at 15 and 50 mg/kg/day was statistically significantly higher compared to controls on day 15 of treatment. A statistically significant increase in body weight gain was observed on postpartum day 8 for animals of the low and mid-dose groups and on post-partum day 12 for the high-dose group compared to controls. A statistically significant reduction in body weight gain was observed in high-dose females on post-partum Day 0 and was considered to be related to the higher number of pups compared to controls. These occasional differences are not considered to be of toxicological significance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of the F0 generation was unaffected by treatment

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

A total of three males, one in the control, one in the mid-dose and one in the high-dose group failed to induce pregnancy.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

All dams gave birth between days 21 or 22 post-coitum, with the exception of one low-dose female which gave birth prematurely on gestation day 18. This isolated case was considered incidental. The statistically significant reduction observed in gestation periods for high-dose females compared to controls was not considered to be of toxicological significance

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no differences in concentration (million sperm/gr epididymal cauda), viability, motility and morphology of the sperm between the control, the high-dose males and in males which failed to induce pregnancy.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no differences between the controls and the treated groups in litter size, litter weight, mean pup weight or pup loss throughout the whole lactation period. Lactation index was similar between the control and the treated groups.
Sex ratio of Fl pups: sex ratios of offspring at birth and on Day 21 post-partum did not show any differences between groups. The statistically significant difference in the number of male pups found dead during the weaning period was attributable to the low number of deaths in the high-dose litters compared to controls and was not considered to be a sex related mortality.

Effect levels (P0)

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity, fertility

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects up to this dose group

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The abnormalities observed in pups during the post-partum period were incidental with no relation to treatment.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no differences between the controls and the treated groups in litter size or pup loss throughout the whole lactation period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no differences between the controls and the treated groups in litter weight or mean pup weight throughout the whole lactation period.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No signs of toxicological significance were observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment related changes were seen in FI pups which died before weaning. No findings of toxicological significance were seen at the necropsy performed on F1 culled pups. There were no meaningful differences in the incidence of the abnormalities recorded at the necropsy of FI pups at weaning or in selected pups.

Description (incidence and severity):

Pre-weaning development of F1 pups: there were no significant differences between groups in the results obtained from the parameters used to monitor the pre-weaning physical and functional development. One pup in group 2 had bilateral anophthalmia (considered incidental) and therefore was not subjected to the pupil reflex test. Values of eye opening and pupil reflex for this litter were excluded from group mean calculation.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

Postnatal development (up to weaning at postnatal day 21)

Generation:

F1

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects up to this dose group

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Reproduction parameters

Reproduction parameters		Test groups (mg/kg bw)
		0	15	50	200
Females	Initial group size	24	24	24	24
	Not pregnant	1	0	1	1
	Total litter loss	0	0	1	0
	With live pups at weaning	23	24	22	23
	Pre-coital interval (days)	4.5	3.75	3.17	2.96
	Copulation index (%)	100	100	100	100
	Fertility index (%)	95.8	100	95.8	95.8
Males	Initial group size	24	24	24	24
	Found dead	1	0	0	0
	Humane kill	1	0	0	0
	Failed to induce pregnancy	1	0	1	1
	Copulation index (%)	100	100	100	100
	Fertility index (%)	95.8	100	95.8	95.8

 

Table 2: Implantation and pre-birth loss data from F0 females(group mean data)

Treatment (mg/kg bw)	Total liter size		Implantations/litter		Pre-birth loss /litter				Gestation period (days)
	N	Mean±SD	N	Mean±SD	Number		Percentage		N	Mean±SD
					N	Mean±SD	N	Mean±SD
0	23	14.7±2.6	23	13.6±2.6	23	1.1±1.5	23	7.0±9.6	23	21.9±0.3
15	24	15.1±2.1	24	14.1±2.4	24	1.0±1.1	24	7.0±7.8	23	22.0±0.3
50	22	14.4±2.2	22	13.4±2.6	22	1.0±1.0	22	9.6±11.8	23	21.9±0.3
200	23	14.9±1.8	23	14.0±2.2	23	0.9±0.9	23	6.1±6.3	23	21.6±0.5*
SD: standard deviation; *: p<0.05

 

Applicant's summary and conclusion

Conclusions:

Oral administration with the test item to parental F0 animals, prior to pairing and throughout gestation and lactation periods at dosages of 15, 50 and 200 mg/kg/day showed no signs of toxicological significance. Reproductive function, as assessed by oestrus cycles, mating performance, pregnancy rate and parturition was not affected by treatment. These results suggest that a dosage of 200 mg/kg/day can be considered the NOAEL.

Executive summary:

The effects of the test article on reproductive function and fertility were evaluated in male and female rats following oral administration by gavage at dosages of 15, 50 and 200 mg/kg/day. Male parental (F0) rats were treated for ten weeks prior to pairing until the day before sacrifice. Female parental (F0) rats were treated for two weeks prior to pairing, during gestation and lactation periods and up to the day before sacrifice, on or shortly after day 21 post-partum. A concurrent group received the vehicle (peanut oil) at the same dose volume and acted as a control. Each group was comprised of 24 males and 24 females.

There were no mortalities in the treated groups. Two control males died (one was found dead and another was humanely sacrificed) during the course of the study. A total of 3 males failed to induce pregnancy; one each in the control, mid and high-dose groups and consequently the females in the same groups were not pregnant. One mid-dose female had total litter loss on post-partum day 0 and one low-dose female gave birth prematurely on gestation day 18. No treatment-related effects were observed at the post-dose observations. There were no treatment-related clinical signs. There were no changes of toxicological significance in body weight or body weight gain in F0 males. The occasional differences observed in F0 females are not considered to be of toxicological significance. Food consumption of the F0 generation was unaffected by treatment. Oestrus cycle, mating performance, and pregnancy rate were unaffected by treatment. No effects on implantation, pre-birth loss data or gestation length were seen in F0 treated females. Total and live litter size, litter and mean pup weights and pup loss were similar between groups. Sex ratios and lactation index were not affected by treatment. No signs were observed during the post-partum period which could be considered related to maternal treatment. Pre-weaning development did not show any differences between the control and the treated groups. There were no toxicologically relevant necropsy findings in Fl pups which died during the lactation period or in Fl pups which were culled on Day 4 post-partum. No changes were seen at the necropsy of Fl pups sacrificed on or shortly after Day 21 post-partum. No findings of toxicological significance were seen at necropsy of Fl selected pups. No signs of toxicological significance were observed at necropsy of the F0 generation. There were no differences in sperm motility or sperm concentration between the control, the high-dose group or the mid-dose male which failed to induce pregnancy.

In conclusion, oral administration with the test item to parental F0 animals, prior to pairing and throughout gestation and lactation periods at dosages of 15, 50 and 200 mg/kg/day showed no signs of toxicological significance. Reproductive function, as assessed by oestrus cycles, mating performance, pregnancy rate and parturition was not affected by treatment. These results suggest that a dosage of 200 mg/kg/day can be considered the NOAEL.