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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987 - 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(May 12, 1981)

Deviations:

no

Remarks:

No treatment during gestation days 16-18, as this was not required in the OECD testing guideline 414 version valid at that time. Amount of vehicle higher than recommended (0.5ml instead of 0.4 ml/100g bw/day).

GLP compliance:

yes

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: room temperature
- Physical state: liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, 8741 Sulzfeld, West Germany
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 180 to 245 g
- Housing: Prior to mating and during mating, the female rats were housed in groups of twenty to twenty-five in communal cages.Mated female rats were individually housed in solid floor macrolone cages of type II with stainless steel lids (dimensions: 260 mm x 200 mm x 140 mm; E. Becker & Co GmbH, 4620 Castrop-Rauxel, West Germany).
- Diet: ad libitium
- Water: ad libitium
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 40 to 70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19.03.1987 To: 23.04.1987 (date of last necropsy)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Solubility of the test item
- Concentration in vehicle: 0, 10, 30 and 60 mg/ml
- Amount of vehicle (if gavage): 5ml/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for analysis were taken from dose formulations prepared for each dose level on 27.03.1987 (beginning of treatment), deep-frozen immediately after formulation, and sent to the study sponsor for analysis in dry ice, inadvertently not before 15.07.1987. Based on the analytical data, it was concluded that the test article was sufficiently stable during the application period.

Details on mating procedure:

The male and female animals were mated at a ratio of 1 : 4 in communal cages during the night.
The females were examined on the following morning for the presence of sperm and/or a vaginal plug.
The day on which sperm and/or a vaginal plug were observed was designated day 0 of gestation.

Duration of treatment / exposure:

Day 6 to 15 post-coitum (inclusive)

Frequency of treatment:

daily

Duration of test:

Day 6- 15 with treatment, maintained without treatment until day 20 post-coitum and were sacrificed and examined on that day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Based on the results of a screening study (Report no. 614-380/42,dated March 1986)

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations :signs of ill-health, toxicity, behavioural change, mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: see above

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each inseminated female rat was recorded on days 0, 6 to 15, and 20 post-coitum and evaluated for days 0, 6, 10, 15, and 20 postcoitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
The food consumption of each inseminated female rat was recorded for days 0 to 6, 6 to 10, 10 to 15, and 15 to 20 post-coitum.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: any abnormalities were recorded.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Individual foetal weights and sex of the foetuses

Intra-uterine deaths were classified as follows:
Early resorptions showed decidual or placental tissues only.
Late resorptions showed embryonic or foetal tissue in addition to placental tissue but excluded foetuses dying in utero within approximately two days
prior to the terminal kill. Dead foetuses included only the foetuses dying in utero within approximately the last two days.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: see below

Half of the foetuses from each litter (taking every second foetus in accordance with the position in the uterine horn, if possible) were eviscerated
and preserved in 95 per cent ethanol for determination of skeletal abnormalities (Alizarin staining technique).
The remaining half was fixed in Bouin's fixative for determination of vis ceral abnormalities (Wilson technique).
The uteri of apparently non-pregnant females were immersed in a 10 per cent solution of ammonium sulphide to reveal evidence of implantation (Salewski technique).

Structural deviations were classified as:
Malformations: rare and/or probably lethal e.g. hydrocephaly.
Variations: changes which regularly occur also in control groups and which are not of functional significance.

Statistics:

For body weight, body weight gain, food consumption, and mean foetal weight (overall, males, females) the analysis of variance was performed with one factor TREATMENT followed by the Newman-Keuls test for multiple group comparison. Number of corpora lutea, number of implantation, number of foetuses, preimplantation loss, post-implantation loss, and proportion of male foetuses were statistically analysed using the Kruskal-Wallis-test.
In case of suspected significance (probability > chi square < 0.05), the four groups were compared two by two using the Wilcoxon two-sample test (normal approximation - with continuity correction of 0.5).
All significances found (at least p < 0.05) are indicated in the respective tables.
The statistical evaluation was performed with the standard software package SAS release 6.02.

Indices:

pregnancy indices
pre-implantation loss
post-implantation loss
sex ratio

Historical control data:

Historical control data is included in the report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no behavioural changes and no clinical observations in any animal of all groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In group 4 (300 mg/kg) body weight gain during gestation, particularly during late gestation, was reduced (16 % less than the control group from day 10 to 15 post-coitum, and 8 % less from day 15 to 20 post-coitum). The difference in comparison with the control group was not statistically significant. This reduced body weight gain is considered to be related to treatment with the test item. Body weight gain of groups 2 (50 mg/kg) and 3 (150 mg/kg) was comparable with the concurrent control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean daily food consumption was slightly lower in group 4 (300 mg/kg) than in the control group, particularly during treatment. In groups 2 (50 mg/kg) and 3 (150 mg/kg) mean daily food consumption was
comparable with the control group.

Description (incidence and severity):

At necropsy unilateral dilatation of the renal pelvis was found in one female of group 3 (150 mg/kg) and two females of group 4 (300 mg/kg). The nature of these findings is considered not to be treatment-related.

Maternal developmental toxicity

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Pre-implantation loss was high in the control group and in group 4 (300 mg/kg). As pre-implantation loss of group 4 (300 mg/kg) was only slightly higher than in the control group (which remains in the upper range obtained from historical background data, this finding is considered to be incidental. In groups 2 (50 mg/kg) and 3 (150 mg/kg) there was no effect on pre-implantation loss. There was no effect of treatment on post-implantation loss, post-implantation loss was lowest in group 4 (300 mg/kg).

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There was no effect of treatment on pregnancy incidence

Effect levels (maternal animals)

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean foetal weight was slightly increased in group 4 (300 mg/kg). Differences to the control group were statistically significant. This finding must be considered in relation to the reduced mean number of foetuses per female in the highest dose group and not in relation to treatment. Mean foetal weights of groups 2 (50 mg/kg) and 3 (150 mg/kg) were comparable with the control group.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of foetuses per dam was slightly lower in group 4 (300 mg/kg). This finding is considered to be related with the slightly reduced mean number of implantations per female and not considered to treatment-related. The mean number of foetuses per dam in groups 2 (50 mg/kg) and 3 (150 mg/kg) was comparable with the control group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex distribution of foetuses did not reveal any compound-related effect.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

An external malformation as micrognathia was found in one foetus of group 2 (50 mg/kg). A further foetus in a second litter of the same dose group showed bilateral anophthalmia. In group 3 (150 mg/kg), one foetus with apodia was observed. In group 4 (300 mg/kg) as well as in the control group no malformations were found.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Skeletal malformations were not detected. The incidence of skeletal variations did not reveal any treatment-related effects.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no efffects

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1: Uterine and Implantation data

		Control	50 mg/kg	150 mg/kg	300 mg/kg
		n=24	n=21	n=20	n=20
Corpora Lutea	total	396	354	323	278
	mean	16.5	16.9	16.1	13.9
Implantations	total	296	289	267	202
	mean	12.3	13.8	13.4	10.1
% Pre-Implantation loss		24.0	16.5	14.6	28.8
Live Foetuses	total	277	273	250	193
	mean	11.5	13.0	12.5	9.7
	% of implantations	93.3	94.8	93.5	96.5
Early resorptions	total	18	16	16	9
	mean	0.8	0.8	0.8	0.5
late resorptions	total	1	0	0	0
	mean	0.0	0.0	0.0	0.0
Dead foetuses	total	0	0	1	0
	mean	0.0	0.0	0.1	0.0
Total intrauterine deaths	total	19	16	17	9
	mean	0.8	0.8	0.9	0.5
% Post-Implantation loss		6.1	5.2	6.5	3.5

Table 2: Group Mean Body weight (g)

calculated from animals with live fetuses

Day of gestation	control	50 mg/kg	150 mg/kg	300 mg/kg
0	206.5	204.3	207.3	200.8
6	241.9	239.5	243.5	234.5
10	260.6	257.9	260	252.5
15	290.4	288.6	289.3	277.5
20	345.2	350.2	350.5	327.8

Table 3: Body weight gain (g) for different gestation stages (data for dams with live offspring)
	days 0-6	days 6-10	days 10-15	days 15-20*
control	35	18.8	29.8	54.8
SD	5.7	8.8	6.2	13.9
50mg/kg bw	35.2	18.3	30.7	61.7
SD	7.3	8.4	5.8	9.9
150 mg/kg bw	36.3	16.5	29.3	61.3
SD	8.1	7.3	5.9	9.4
300 mg/kg bw	33.8	18	25	50.3
SD	7	7.3	9.3	21
*Last day of treatment was day 15
SD = Standard Deviation

Table 4: fetal data

	control (n=24)	50 mg/kg (n= 21)	150 mg/kg (n= 20)	300 mg/kg (n= 20)
Number of fetuses	277	273	250	193
mean number of fetuses per female	11.5	13	12.5	9.7
mean litter weight (g)	38.7	43.2	42.6	33.5
mean fetal weight (g) overall	3.33	3.33	3.39	3.51
mean fetal weight (g) males	3.42	3.42	3.51	3.53
mean fetal weight (g) females	3.23	3.25	3.27	3.46
number of males	147	126	124	94
number of females	130	147	126	99
sex ratio in % (males : females)	53.1 : 46.9	46.2 : 53.8	49.6 : 50.4	48.7 : 51.3

Table 5: Malformation, variation data

	control	50 mg/kg	150 mg/kg	300 mg/kg
Number of fetuses examined externally	277	273	250	193
Number of fetuses with external malformations	0	1	1	0
% of fetuses with external malformations	0	0.4	0.4	0
Number of litters with external malformations	0	1	1	0
Number of fetuses examined viscerally	144	141	126	102
Number of fetuses with visceral malformations	0	1	0	0
% of fetuses with visceral malformations	0	0.7	0	0
Number of litters with visceral malformations	0	1	0	0
Number of fetuses examined viscerally	144	141	126	102
Number of fetuses with visceral variations	1	1	0	0
% of fetuses with visceral variations	0.7	0.7	0	0
Number of litters with visceral variations	1	1	0	0
Number of fetuses examined skeletally	133	132	124	91
Number of fetuses with skeletal malformations	0	0	0	0
% of fetuses with skeletal malformations	0	0	0	0
Number of litters with skeletal malformations	0	0	0	0
Number of fetuses examined skeletally	133	132	124	91
Number of fetuses with skeletal variations	133	132	123	91
% of fetuses with skeletal variations	100	100	99.2	100
Number of litters with skeletal variations	24	21	20	18
Total Number of malformed fetuses	0	2	1	0
Total number of litters with malformed fetuses	0	2	1	0
Average % malformed fetuses	0	0.7	0.4	0

Applicant's summary and conclusion

Conclusions:

In conclusion, administration of the test substance during organogenesis by oral gavage at a dose level of 300 mg/kg elicited slight maternal toxicity (16% reduction in body weight gain and - possibly - slightly reduced food consumption), but no embryotoxicity, and no teratogenicity. Administration of 50 and 150 mg/kg did not elicit any maternal toxicity, embryotoxicity, or teratogenicity.

Executive summary:

Groups of 25 sexually mature and mated female rats received the test item by oral gavage at dosages of 50, 150, or 300 mg/kg daily for ten consecutive days from day 6 to 15 post-coitum, inclusive. A further group of 25 rats of the same strain which received the vehicle (arachis oil) over the same period served as the control group. The animals were sacrificed on day 20 post-coitum. After conclusion of the present study with including the evaluation of the data, the results of the study can be summarized as follows:

There were no clinical observations and no necropsy findings which might be considered to be related to treatment. At a dose level of 300 mg/kg, maternal body weight gain during gestation was slightly reduced. This is considered to be a slight maternally toxic effect of the compound. Mean daily food consumption was slightly lower in group 4 (300 mg/kg) than in the control group. This finding might be a slightly toxic effect of treatment. There was no effect of treatment on pregnancy incidence. There was no effect of treatment on implantations. Post-implantation loss was not affected by the test item. The mean number of foetuses per dam was slightly reduced in group 4 (300 mg/kg). This finding is considered to be related with the slightly reduced mean number of implantations per female and not to be a compound-related effect. Sex distribution was not affected by treatment. Mean foetal weight of group 4 (300 mg/kg) was slightly increased. This finding is considered to be related with the slightly reduced mean number of foetuses per female in this group and not to indicate any effect of treatment. Nature and incidence of malformations and variations did not reveal any compound-related effect.

In conclusion, administration of the test article during organogenesis by oral gavage at a dose level of 300 mg/kg elicited slight maternal toxicity (slightly reduced body weight gain and - possibly - slightly reduced food consumption), but no embryotoxicity, and no teratogenicity. Administration of 50 and 150 mg/kg did not elicit any maternal toxicity, embryotoxicity, or teratogenicity.