Pentyl salicylate

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

not specified

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study according to OECD TG 415 with restrictions. To address toxicological endpoints as part of the REACH registration of Benzyl Salicylate (Target Substance) it is proposed to read-across to Cyclohexyl Salicylate (Source Substance). The use of read-across works within the spirit of REACH and the stated aim of the legislation to reduce animal testing where possible. The Target Substance and Source Substance have been characterised using the categories and databases present in the OECD [Q]SAR Toolbox. From the profiling, it can be seen that the two substances share structural similarities and also ‘mechanistic action’ similarities which are both general and endpoint specific. Therefore, read across is justified.

Justification for type of information:

The justification for read across from Cyclohexyl Salicylate is attached in section 13.2 (Other assesment report).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

as of 26 May 1983

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

25485-88-5

EC Number:

607-733-0

IUPAC Name:

25485-88-5

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Cyclohexylsalicylat
- Physical state: colourless liquid
- Analytical purity: not reported
- Impurities (identity and concentrations): not reported
- Composition of test material, percentage of components: not reported
- Lot/batch No.: 11183334/2
- Expiration date of the lot/batch: December 1995
- Storage condition of test material: at ambient temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sulzfeld, Germany
- Age at study initiation: (P): males were aged about 16 weeks and females about 10 weeks at initiation of mating
- Weight at study initiation: males weighed 172 to 262 g and females 178 to 221 g
- Housing: animals were housed individually in Makrolon cages (except during mating and lactation) with Lignocel 3-4-Fasern softwood bedding
- Diet (e.g. ad libitum): Ssniff R-Z pelleted diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: males were acclimatised for 13 days and females for 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 ± 1.5 °C
- Humidity (%): 40 to 70
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

IN-LIFE DATES: From: 4 October 1994 To: 2 February 1995

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Solutions of test substance in corn oil were prepared freshly every second day by means of a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no justification given
- Concentration in vehicle: depending on the dose level the concentrations of test substance in corn oil were 12, 36 or 108 g/L, respectively
- Amount of vehicle (if gavage): a volume of 5 mL/kg was administered to test animals

Details on mating procedure:

- M/F ratio per cage: animals were mated monogamously
- Length of cohabitation: males and females were housed overnight during a mating period of 21 days
- Proof of pregnancy: the finding of vaginal plug or sperm in vaginal smear was defined as day 0 of gestation
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

An analysis of the dosing solutions was carried out at commencement, during the 10 weeks of administration and at termination of the treatment. The measured concentrations agreed reasonably well with the nominal values. Analysis was performed by HPLC with a LiChrosorb RP18 column (250 x 4 mm, 5 µm) and Acetonitrile/water (70/30) or Acetonitrile as mobile phase, a flow rate of 1 mL/min, 10 µL injection volume and UV detection at 223 nm.

Duration of treatment / exposure:

Males were treated for 10 weeks before mating and throughout mating up to sacrifice (i.e. 14 weeks of treatment)
Females were treated for 2 weeks before mating, throughout mating, gestation and lactation up to weaning or day 21 post partum

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
60 mg/kg bw
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
180 mg/kg bw
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
540 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

24 males and 24 females in each dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen on the basis of information generated in the 90-day oral repeated dose toxicity study.

Positive control:

not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: weekly in males, weekly in females except during gestation and lactation where daily weighing was performed

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Frequency: see body weight examination

MATING PERFORMANCE
- Frequeny: vaginal smears were taken daily during the mating period until successful mating was confirmed and the number of days until successful mating was recorded.

GESTATION PERIOD
The gestation period of dams with litter was taken as the time between the day of successful mating and the day of birth.

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

Parameters examined in male parental generations:
Weights of testes, epididymides, seminal vesicles with coagulating gland and prostate were determined

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no data

PARAMETERS EXAMINED

The following parameters were examined in offspring during general observations: number of alive and dead pups, determination of sex, weight, external abnormalities

The following parameters were examined in offspring to assess pre-weaning development: pinna unfolding, hair growth, eye opening, upper incisor eruption, testis descent was assessed in males once at weaning, surface righting reflex from day 1 post partum to 100% success within litter, auditory startle refle from day 12 post partum to 100% success within litter, air righting reflex once between day 18 and 21 post partum, pupil reflex once between day 18 and 21 post partum

The following tests were made with at least 10 male and 10 female pubs in each dose group between day 18 and 21 post partum: inclined plane test according to Rivlin and Tator (1977, J. Neurosurg. 47, 577-581), open field test according to Candland and Campbell (1962, J. Comp. Physiol. Psychol. 55, 593-596)

GROSS EXAMINATION OF DEAD PUPS: yes

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: males were sacrificed after 14 weeks of treatment
- Maternal animals: parent females were sacrificed on or shortly after day 21 of lactation or after death of the whole litter

GROSS NECROPSY
- Gross necropsy of male parents consisted of macroscopic examination of internal viscera, determination of weight of testes, epididymides, seminal vesicles with coagulation gland and prostate and liver. Inspection of pituary gland, liver and tissues showing severe changes was performed (all these organs were preserved)
- Gross necropsy of female parents consisted of inspection of the uterus for implantation sites, determination of liver weight, ovaries, uterus, cervix, vagina, pituary gland, liver and tissues showing severe changes (all these organs were preserved in 8% formalin)

HISTOPATHOLOGY
No data

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed on or shortly after day 21 post partum

GROSS NECROPSY
- The pups were subjected to postmortem external and internal examination for abnormalities

HISTOPATHOLOGY / ORGAN WEIGTHS
Two male and female pups were selected from each litter in order to determine the weights of brain, heart, liver, kidneys and spleen. Tissues showing severe changes were preserved at the discretion of the post mortem pathologist.

Statistics:

Body weight changes, food consumption, number of implantations and offspring were analysed for variance with a subsequent multiple range tes, or, if indicated, group mean values were compared by the Kruskal-Wallis test and the Mann-Whitney test. Mean body weights, food conversion, litter and pup weights were compared by the Dunnett test. Organ weights were evaluated both as absolute and relative weights and values found in the dose groups were compared to those in the control group by the two-tailed Dunnett test modified according to Kramer. Sex distribution, gestation length, number of days until successful mating, physical and reflex development were analysed by the Kruskal-Wallis test and the Mann-Whitney U-test. Behaviour in the inclined plane and open field tests were analysed by the Kruskal-Wallis test and the Wilcoxon 2-sample test (normal approximation).

Reproductive indices:

Mating index (no. of "positive" females for which mating was confirmed/no. of paired females * 100), fertility index (no. of pregnant females/no. of paired females *100), conception rate (no. of pregnant females/no of "positive" females * 100), abortion rate (no. of dams showing abortion/no. of pregnant dams * 100)

Offspring viability indices:

Gestation index (no. of litters with live foetuses/no. of pregnant females * 100), pre-birth loss index ([no. of implantations - total litter size]/no. of implantations * 100), pup loss index (at birth) ([total litter size at birth - live litter size at birth]/total litter size at birth * 100), cumulative pup loss index (day x) ([total litter size at birth - live litter size on day x]/total litter size at birth * 100), sex ratio (no. of lie male pups per litter/no. of live pups per litter * 100)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): one male of the 180 mg/kg and one male of the 540 mg/kg groups died during the treatment: one animal was killed in extremis following probably false administration, one animal was found dead in the pre-mating phases and necropsy of this animal did not reveal any reason of death.
Besides the increase in the relative liver weight, Ia distinct lobular pattern of the liver seemed to be increased in females of the high dose group. A number of treatment related findings in females of the high dose group were seen in the third week of gestation, particularly on the day of delivery. These effects included reduced activity, rough fur and shutting of the eyes. In addition, ataxia, stretched body posture, respiratory disorder and poor general condition were observed in two females which died during delivery. Five females in the high dose group had an increased amount of blood in the bedding during delivery. All these findings were indicative of dystocia. One female in the high dose group showed no care of pubs during and after birth. No females showed signs of abortion or premature delivery.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): weight gain was significantly reduced in the males of the high dose group in the second half of the pre-mating phase. Also food conversion was significantly affected in males. Weight gain in females of the high dose group was significantly decreased during the third week of gestation. Weight gain of females in the high dose group was significantly increased during the entire period of lactation although mean food consumption values were decreased in these females.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): not applicable as substance was administered by oral gavage

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): no data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): Examination of male reproductive organs revealed no differences between treated groups and controls

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): No differences between treated groups and controls were found.

ORGAN WEIGHTS (PARENTAL ANIMALS): Relative liver weights were significantly increased in males of the high dose group. Absolute and relative liver weights were significantly increased in females of the high dose group.

GROSS PATHOLOGY (PARENTAL ANIMALS): Reddening of mucous membrane of the stomach, which was accompanied by a white coating, was noted in females of the high dose group at a higher incidence.

HISTOPATHOLOGY (PARENTAL ANIMALS)

OTHER FINDINGS (PARENTAL ANIMALS):

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: General toxicological effects

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: General toxicological effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING): The total and live litter size at birth was significantly reduced in the high dose group. Live litter size was also significantly reduced on days 4, 7, 14 and 21 post partum in the high dose group. The number of litters with dead pups at birth was slightly increased in the high dose group. Cumulative pup loss indices were significantly increased in the high dose group from day 4 post partum onwards. The ratio of litters with dead pups to litters without dead pups up to day 21 post partum in the high dose group differed significantly from that of the controls and the number of litters with 4 or more pup losses from birth to day 21 post partum was also increased in the high dose group. Moreover, total litter death occurred in two litters in the high dose group.

CLINICAL SIGNS (OFFSPRING): The number of litters and pups showing haematoms up to day 10 post partum was slightly increased in the high dose group. This finding possibly corresponds to the signs of dystocia.

BODY WEIGHT (OFFSPRING): mean group body weights of male and female pups were significantly reduced in litters of the high dose group after birth and in females also on day 4 post partum. Litter weights were significantly decreased in the high dose group from birth to day 21 post partum.

SEXUAL MATURATION (OFFSPRING): no data

ORGAN WEIGHTS (OFFSPRING): a significant increas in relative heart weight was seen in male pups in the high dose group, whereas the weights did not differ significantly in female pups of this group. Absolute and relative liver weights were significantly decreased in male pups in the groups treated with 60 or 540 mg/kg bw and in female pups in the high dose group. Relative liver weights in female pups in the group treated with 180 mg/kg bw were also found. No clear dose-response relationship was seen in males.

GROSS PATHOLOGY (OFFSPRING): necropsy of pups found dead during the pre-weaning period did not reveal any specific findings which point to a manifestation of a parental treatment effect. Macroscopic examination of pups sacrificed on day 21 post partum revealed an increased number of litters with pups showing a truncated coniform heart (2 male pups and 4 female pups in the high dose group)

HISTOPATHOLOGY (OFFSPRING)

OTHER FINDINGS (OFFSPRING): physical development was retarded in the high dose group. A significantly decreased frequency of pups showing testis descent at weaning in the group receiving 180 mg/kg bw was considered incidental as no dose relationship existed. Onset of the auditory startle reflex was significantly retarded in the pups from the high dose group. No significant differences of pup behaviour in the different groups were found in the inclined plane test.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental effects

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Repeated oral administration of the substance by gavage to male and female Wistar rats during gametogenesis, mating, gestation and lactation up to 21 days post partum had no adverse effects on reproduction at a dose of 180 mg/kg/day in females and 540 mg/kg/day in males.

Executive summary:

Potential adverse effects of the test substance cyclohexyl salicylate on the reproduction of male and female rats and on their offspring resulting from daily oral administration to the parent animals during gametogenesis, mating, gestation and lactation up to day 21 post partum was studied in a one-generation study under GLP in accordance with OECD TG 415 as of May 1983. Four groups of 24 female and 24 male rats each were used in the study. The substance was dissolved in corn oil and administered to parent animals by oral gavage daily at a constant volume of 5 mL/kg body weighat doses of 0 (vehicle control), 60, 180 and 540 mg/kg bw in these groups. Evaluation of indices of mating performance and fertility (mating index and performance, fertility index and conception rate) showed no difference between the treated groups and the control group. Examination of male reproductive organs revealed no differences between treated groups and controls. During necropsy of males and females no distinctly treatment related findings were noted. However, liver weights were significantly increased in the high dose group. In addition, a distinct lobular pattern of the liver seemed to be increased in females of the high dose group. A number of findings were noted in female parent animals of the high dose group at the end of gestation, particularly at the day of delivery. These findings comprised reduced activity, shutting of eyes, respiratory disorders and increased amount of blood in the bedding during delivery and were interpreted as signs of dystocia. Additionally, two pre-terminal deaths occurred shortly before or during delivery in the high dose group. High dose females showed significantly decreased body weight gain during gestation, especially in the last week of gestation. No females showed signs of abortion or premature delivery throughout pregnancy. Evaluation of gestation index, gestation length and number of implantations provided no evidence of a treatment releated effect. A slight effect on pre-birth loss was found in the high dose group receiving 540 mg/kg bw/day. The litter size was significantly reduced in the high dose group from birth to day 21 post partum, whereby the sex ratio did not show any important differences. Pup loss at birth was slightly increased in the high dose group. Cumulative pup loss was significantly increased in this group from day 4 post partum, also becoming evident as an increased number of litters with 4 or more pup losses during pre-weaning development. Treatment-related retardation in pinna unfolding, upper incisor eruption, eye opening and onset of the auditory startle reflex was observed in pups of the high dose group. Mean group body weights of the pups at birth were significantly reduced in the high dose group. Necropsy of pups on day 21 post partum gave a higher number of litters with pups showing a truncated coniform heart in the high dose group. Additionally, the weights of heart, liver and brain seemed to be affected in the pups of the high dose group. It was concluded that the NOAEL for reproductive effects was 180 mg/kg bw/day in female rats and 540 mg/kg bw/day in male rats. The NOAEL for developmental effects in the pups was 180 mg/kg bw/day.