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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
From 1984/03/16 to 1982/12/23.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Justification for type of information:
A category approach is proposed for hexyl salicylate using data from other salicylate substances. The members of this category are demonstrated to be metabolised rapidly by esterases to salicylic acid; systemic exposure will therefore be to the metabolites to a much greater extent. The systemic toxicity of the salicylates is comparable and is attributable to the salicylic acid generated by metabolism. The other product of metabolism is the alcohol liberated from the sidechain following esterase metabolism. In the case of hexyl salicylate, this alcohol will be 1-hexanol which is of low toxicity and is rapidly metabolised and excreted and/or incorporated into normal metabolism. Hexanol is of lower toxicity than other alcohols generated from the metabolism of other category members (e.g. methanol); the source data therefore represents a worst case approach.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report Date:
1984

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: US NTP continuous breeding protocol
Principles of method if other than guideline:
The study design involves co-housing for a prolonged period.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: oily liquid
Details on test material:
- Name of test material: methyl salicylate (MeS)
- Molecular Formula: C8H8O3
- Molecular Weight: 152.14
- Physical state: oily liquid
- Analytical purity: ≥ 99%
- Impurities (identity and concentrations): no data
- Lot/batch No: Lot No. 703535, Batch No. 01
- Stability under test conditions: MeS is stable as the bulk chemical when stored protected from light for 2 weeks at temperatures up to 60°C.
- Storage condition of test material: the material was stored for 2 weeks at temperatures of -20, 5, 25 and 60°C in amber glass septum vials sealed with Teflon-lined septa.
- Source: Fisher Scientific Co.

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory, Inc. (Kingston, NY).
- Age at study initiation: eleven- week old.
- Weight at study initiation: 35.6 +/- 0.76 for males and 26.3 +/- 0.66 for females.
- Fasting period before study: no data
- Housing: 2 animals per cage.
- Diet: ad libitum (purina certified pelleted Rodent chow)
- Water: ad libitum
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (C°): between 20-25 °C
- Humidity (%): between 20 to 70%
- Air changes (per hr): at least 10 or more air changes per hour of HEPA-filtered air.
- Photoperiod (hrs dark / hrs light): 10 hrs dark/14 hrs light.

IN-LIFE DATES: no data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:


DIET PREPARATION
- Rate of preparation of diet (frequency): every two weeks
- Mixing appropriate amounts with (Type of food): dosing solutions were formulated by mixing the test article directly into different proportions of corn oil.
- Storage temperature of food: Each formulated sample will be refrigerated prior to and between dosing. An aliquot will be removed for dosing and warmed to room temperature.


VEHICLE: corn oil
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): no data
- Purity: no data
- Source: Mazola.
Details on mating procedure:
- M/F ratio per cage:
1- For Task 2: one breeding pair will be housed per cage: 40 breeding pairs in the control group, and 20 breeding pairs per group for the three groups receiving test chemical.
2- For Task 3: one male mice, previously exposed to the MTD of the test article will be matched with control female mice, similarly one female mice previously treated with the MTD of the test article will be matched with control males. The remaining 20 control males will be randomly matched with the remaining 20 control females.
- Length of cohabitation:
1- For Task 2: 100 days
2- For Task 3: 7 days

- Proof of pregnancy: vaginal plugs.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how):
- Task 2: after 14 weeks of cohabitation, each pregnant female was isolated, housed individullay for 3 weeks.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Aliquots of various dosage formulations were sent to MRI for chemical analysis. reference aliquots were within 93 to 102 percent of the indicated MeS concentrations. These limits were considered acceptable. (The detailed reports describing analysis of various reference samples are present in Appendix III of NTP report)
Duration of treatment / exposure:
7 days prior to mating then for 14 weeks of cohabitation period and 3 weeks thereafter.
Frequency of treatment:
daily
Details on study schedule:
not performed
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
- 20/sex/dose for MeS groups
- 40/sex/dose for vehicle control group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: prior to commencing the reproductive study, a preliminary range-finding study was performed. The maximum tolerated dose (MTD) in male and female CD-1 mice was determined.

Positive control:
Not required for this study type.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: yes


DETAILED CLINICAL OBSERVATIONS: no

BODY WEIGHT: yes

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no


Oestrous cyclicity (parental animals):
not performed
Sperm parameters (parental animals):
not performed.
Litter observations:
not performed.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, after Task 3.

- Maternal animals: All surviving animals, after Task 3.


GROSS NECROPSY: no


HISTOPATHOLOGY / ORGAN WEIGHTS: not performed.
Postmortem examinations (offspring):
not performed.
Statistics:
Analyses of variance, student-test, determination of P values and non parametric test (Kruskal-Wellis).
Reproductive indices:
- Fertility Index (%): No. Fertile/No. Cohabited X 100.
- Mating Index (%): No. with Copulatory plugs/ No. Cohabited X 100.
Offspring viability indices:
not performed

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs of toxicity.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was no treatment-related mortality
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

No effects of treatment were observed

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
- There was a significant decrease (p<0.05) in the mean number of litters at 500 mg/kg bw/d
- The average number of pups per litter, the proportion of pups born alive, and mean live pup weight values were also significantly reduced in this group compared to the corresponding controls.
- There was no significant effect on any of these parameters in the remaining two dose groups (only a 3% reduction in pup weight at 250 mg/kg).

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Fertility of pairs during continuous breeding

Treatment group No. Fertile/No. cohabited Fertility Index (%)
Control 38/38 100
100 mg/kg bw 17/18 94
250 mg/kg bw 18/18 100
500 mg/kg bw 16/16 100

Applicant's summary and conclusion

Conclusions:
A reproductive NOAEL of 100 mg/kg bw/d is determined for this study, based on reduced litter size and pup weights at 250 and 500 mg/kg bw/d.
Executive summary:

In this NTP study, groups of CD-1 Mice were gavaged with methyl salicylate (in corn oil) at dose levels of 100, 250 or 500 mg/kg bw/d during the 7-day pre-mating period, a 14-week cohabitation period and 3 for weeks thereafter. A significant decrease in the average number of litters and a lower proportion of pups born alive were seen at 500 mg/kg bw/d. Dose-related decreases were found in average litter size and average pup weight with the decreases being statistically significant at 500 mg/kg bw/d and marginally significant at 250 mg/kg bw/d. A reproductive NOAEL of 100 mg/kg bw/d is therefore determined for this study.