Tri-C18-22 (even numbered)-alkyl 2-hydroxypropane-1,2,3-tricarboxylate

Toxicity to reproduction

Administrative data

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Published GLP study conducted according to a recognised guideline

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guidelines for Detection of Toxicity to Reproduction for Medicinal Products

Principles of method if other than guideline:

The study investigated the effects on fertility and reproduction of the substance on adult male and female rats; investigations of developmental toxicity are reported elsewhere.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Behenyl alcohol

IUPAC Name:

Behenyl alcohol

Constituent 2

Reference substance name:

Docosan-1-ol

EC Number:

211-546-6

EC Name:

Docosan-1-ol

Cas Number:

661-19-8

Molecular formula:

C22H46O

IUPAC Name:

docosan-1-ol

Constituent 3

Reference substance name:

221-546-6

IUPAC Name:

221-546-6

Details on test material:

Behenyl alcohol is a saturated 22-carbon, long-chain aliphatic alcohol. The composition and stability of behenyl alcohol was documented throughout the study, no further information is provided.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

The test animals were male and female Sprague-Dawley rats, obtained from Charles River (UK) Ltd., at approximately 5-6 weeks of age. Upon arrival the rats were acclimatised for 6 days prior to dosing. Male rats weighed 193-240 g and were 6-7 weeks old at study initiation. Female rats weighed 208-262 g and were 10-12 weeks old at study initiation. During acclimation and premating, the rats were housed in groups of 5 males and 5 females in suspended stainless steel cages. During the mating period, rats were housed 1 male and 1 female in suspended polypropylene cages with stainless steel mesh lids and floors. During gestation (after mating) the rats were housed in same sex groups of 5 in suspended stainless steel cages. The animal room was maintained at a temperature of 18°C and relative humidity 55% with a 12 hour light-dark cycle. The rats were provided with an expanded rodent diet (Special Diet Services Ltd., England) and tap water ad libitum.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% Tween 80

Details on exposure:

The test substance was administered by gavage in 1% Tween 80 at doses of 0, 10, 100 and 1000 mg/kg bw/d. A constant dosing volume of 5 ml/kg bw was employed. The dose administered was calculated from bodyweights measured immediately before each administration.

Details on mating procedure:

Rats were housed 1 male to 1 female during the mating period. Each morning cage trays were checked for copulation plugs. Vaginal smears were prepared from each female and examined for the presence of spermatozoa. The length of the mating period was recorded (time elapsing between initial paring and detection of mating) and Gestation Day 0 was designated as the day in which evidence of mating was found.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No information available.

Duration of treatment / exposure:

Males were treated for 71 days prior to mating, during mating, and until termination. Females were treated for 15 days prior to mating, during mating, and up to Day 17 of gestation.

Frequency of treatment:

Daily during the treatment period.

Details on study schedule:

Males were treated for 71 days prior to mating, during mating, and until termination. Females were treated for 15 days prior to mating, during mating, and up to Day 17 of gestation. Females were sacrificed on Day 20 of gestation and the uterine contents were examined. Males were sacrificed following necropsy of the females.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 10, 100 and 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

No further details.

Positive control:

Not required for this study type.

Examinations

Parental animals: Observations and examinations:

Cage side observations were made daily to check for signs of toxicity or animals in a moribund condition. A macroscopic examination of the visceral organs was conducted on any animal that died during the study. Prior to mating, food consumption was measured weekly and water consumption was measured daily. During the gestation period (after mating) food and water consumption was measured in females only at the following time points: Gestation Days 0-2, 3-6, 7-9, 10-13, 14-17 and 18-19 inclusive. Male and female bodyweights were recorded twice weekly until mating. Following mating, male bodyweight gains were measured twice weekly for the remainder of the study, and female bodyweight gains were recorded on Gestation Days 0, 3, 7, 10, 14, 18 and 20.

Oestrous cyclicity (parental animals):

Starting 10 days prior to the start of the mating period, vaginal smears were obtained daily from all females to assess the regularity and duration of the oestrus cycle.

Sperm parameters (parental animals):

The left vas deferens was ligated to obtain a 5 µl sample from the cauda epididymis. The sample was then diluted (1/200) in medium M199 + BSA factor V 0.5% w/v, prewarmed to 37°C, and mixed to assess for motility according to the following grades: no sperm motile; few motile sperm; most sperm motile, slow moving; or most sperm motile, fast moving. The number of spermatozoa was assessed using a Fuchs-Rosenthal haemocytometer by further diluting the sample (1/20) in 4% v/v neutral buffered formaldehyde.

Litter observations:

Not performed - dams were sacrificed on Day 20 of gestation.

Postmortem examinations (parental animals):

In addition to examination of the uterine contents, each female was examined macroscopically for evidence of disease or adverse reaction. The number of corpora lutea in each ovary was counted. The reproductive tract including the ovaries was removed and examined for the number of pre- and post-implantation sites, early and late resorptions, viable foetuses, and the distribution of foetuses in each uterine horn. The uterus of any female presumed to be non-pregnant was stained using 10% aq (v/v) ammonium sulphide solution and examined for implantation sites. Placental weights were recorded and examined macroscopically for any abnormalities.
Males were killed following necropsy of the females, and macroscopically examined for abnormalities externally and internally.

Postmortem examinations (offspring):

Each foetus was weighed, subjected to a detailed external examination, and uniquely identified within the litter with respect to the uterine position. The neck, thoracic and abdominal cavities were removed from half the foetuses, the contents of the thoracic and abdominal cavities were examined, and the sex recorded. These foetuses were eviscerated, fixed in methylated spirit, processed and stained with Alizarin Red and subjected to a skeletal examination. The remaining foetuses were placed in Bouin's fixative and internally examined using a modification of the Wilson free-hand serial sectioning technique.

Statistics:

One way ANOVA and t-tests were performed on bodyweights, bodyweight changes, and food and water consumption. Organ weights were evaluated by Dunnett's or Behren's-Fisher's tests. Nested ANOVA and weighed t-test were conducted on foetal and placental weights.

Reproductive indices:

Not determined.

Offspring viability indices:

Not determined.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

All female rats survived to the end of the study. One male 1000 mg/kg bw/d rat was observed with a distended abdomen, pallor, ptosis, irregular respiration and a decrease in bodyweight, and was therefore sacrificed during Week 6. Necropsy revealed watery blood, enlargement of the liver with lobular pattern accentuated, enlarged pale spleen and reduced gastrointestinal tract content. This was the only death that occurred in the study, and none of these findings were observed in other rats of this study (and other studies with behenyl alcohol conducted by the same authors), therefore the death was not considered to be related to treatment. No other clinical signs were observed. There were no effects of treatment on bodyweight gains, food and water consumption, oestrus cyclicity, mating performance or fertility. There were no abnormalities detected at terminal necropsy. There were no effects of treatment on placental weights, the number of corpora lutea, pre- and post-implantation sites, early and late resorptions and viable foetuses.
There were no treatment-related macroscopic findings reported in males. Absolute and relative reproductive organ weights were similar across groups. There were no effects of treatment of sperm number and motility.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects of treatment were seen at the highest dose level

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

There were no treatment-related macroscopic, internal or skeletal variations observed in the foetuses.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects of treatment were seen at the highest dose level

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1. Mean Reproductive Parameters

Parameter	Dose (mg behenyl alcohol/kg bw/d)
	0	10	100	1000
No. pregnant animals	22	22	22	21
Corpora lutea count	17.8 ± 2.7	18.4 ± 4.0	18.7 ± 2.3	18.9 ± 2.4
Implantations	17.2 ± 2.6	17.0 ± 3.2	18.1 ± 1.8	18.0 ± 2.3
Viable young
Male	8.4 ± 2.9	8.4 ± 2.3	8.5 ± 2.5	8.6 ± 2.6
Female	8.0 ± 3.0	7.5 ± 2.6	8.5 ± 2.2	8.3 ± 2.2
Total	16.4 ± 3.2	15.9 ± 3.5	17.0 ± 2.3	16.9 ± 2.2
Resorptions
Early	0.82 ± 0.90	1.09 ± 1.04	1.14 ± 1.07	1.05 ± 1.02
Late	0.00 ± 0.00	0.0 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
Total	0.82 ± 0.90	1.09 ± 1.04	1.14 ± 1.07	1.05 ± 1.02
Implantation loss (%)
Pre	3.3	8.3	3.2	5.8
Post	4.7	6.4	6.3	5.8

Data represent mean ± standard deviation

Applicant's summary and conclusion

Conclusions:

There were no effects of treatment on parental animals, reproductive parameters or foetuses, therefore the NOAEL for reproductive toxicity is considered to 1000 mg/kg bw/d.

Executive summary:

The reproductive toxicity of behenyl alcohol was evaluated in a study with male and female Sprague-Dawley rats. The test material was administered by gavage in 1% Tween 80 at doses of 0, 10, 100 and 1000 mg/kg bw/d. Males were treated for 71 days prior to mating, during mating, and until termination. Females were treated for 15 days prior to mating, during mating, and up to Day 17 of gestation. Females were sacrificed on Day 20 of gestation and the uterine contents were examined. Males were sacrificed following necropsy of the females. One male 1000 mg/kg bw/d male died during the study, but this death was not considered to be treatment related. There were no other deaths during the study. No clinical signs were observed. There were no effects of treatment on bodyweight gains, food and water consumption, oestrus cyclicity, mating performance or fertility. There were no abnormalities detected at terminal necropsy. There were no effects of treatment on placental weights, the number of corpora lutea, pre- and post-implantation sites, early and late resorptions and viable foetuses. There were no treatment-related macroscopic findings reported in males. Absolute and relative reproductive organ weights were similar across groups. There were no effects of treatment of sperm number and motility. There was no evidence that behenyl alcohol is toxic to reproduction in rats, and the NOAEL is considered to be 1000 mg/kg bw/d.