Reaction mass of 3-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde and 4-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

one-generation study

Type of information:

experimental study

Adequacy of study:

key study

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:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 415 (One-Generation Reproduction Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 3-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde and 4-(4-hydroxy-4-methylpentyl)cyclohex-3-enecarbaldehyde

EC Number:

915-617-9

Molecular formula:

C13H22O2

IUPAC Name:

Reaction mass of 3-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde and 4-(4-hydroxy-4-methylpentyl)cyclohex-3-enecarbaldehyde

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, UK
- Age at study initiation: (P) males ca. 6 weeks, females ca. 10 weeks
- Weight at study initiation: (P) Males: 191 - 255 g; Females: 211 - 271 g
- Housing: initially, in groups of 4 in polypropylene cages with stainless steel grid floors and tops. During the mating, animals were housed in similar cages on 1:1 male:female basis. After mating males were transferred to the original cages; females were housed individually during gestation and lactation in polypropylene cages with solid floors and stainless steel lids.
- Diet: pelleted diet (Rodent PMI 5002 (certified) diet, ad libitum
- Water: mains drinking water, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): at least 15/hour
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
- Formulations were prepared weekly

VEHICLE
- Concentration in vehicle: 0, 6.25, 25 and 125 mg/mL
- Amount of vehicle: 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test substance in arachis oil was determined by gas chromatography using an external standard technique.

Duration of treatment / exposure:

76 days pre-mating, maximal 21 days mating, males were killed and examined upon evidence of successful mating, females and offspring were killed and examined on day 21 post-partum. Non-pregnant females were killed and examined after day 25 post-coitum.

Frequency of treatment:

Daily (except for females during littering/parturition)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

Details on mating procedure
- Age at mating of the mated animals in the study: mating was performed on day 76 of treatment.
- M/F ratio per cage: 1:1
- Length of cohabitation: a maximum of 21 days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 1 of pregnancy
- After successful mating each pregnant female was caged individually during gestation and lactation in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes
Dose selection rationale: based on the results of a 14-day dose-range finding study
Rationale for animal assignment: the animals were allocated to dose groups using a randomisation procedure based on stratified body weights.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: the animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before and after the dosing, and 1 and 5 hours post-dosing during the working week. Animals were observed immediately before and after dosing and 1 hour post-dosing at the weekends or public holidays.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, then weekly for males until termination. Females were weighed weekly during maturation and daily during mating. Once mating was evident, body weights were recorded on days 1, 4, 7, 14 and 21 of post-coitum and post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE: yes
- Time schedule for examinations: During the maturation period, weekly food consumption was recorded for each cage. For females showing evidence of mating, food consumption was recorded for the period covering days 1 - 7, 7 - 14 and 14 - 21 post-coitum. For females with live litters, food consumption was recorded for the period covering days 1 - 7, 7 - 14 and 14 - 21 post-partum.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt change.

OTHER:
- During mating, a vaginal smear was prepared for each female daily and the stage of the oestrous cycle was recorded.
- Parameters examined in P male parental generations: testis weight, epididymis weight, numbers of homogenisation resistant spermatids, sperm motility, sperm morphology
- The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, the detachment and unfolding of pinna, incisor eruption and eyelid separation, reflexological response to stimuli by assessing surface righting reflex on Day 1 post-partum and air righting reflex on Day 17 post-partum. Pupillary reflex and auditory startle response were performed on day 21 post-partum.

Sacrifice and pathology:

SACRIFICE
- Male animals: All surviving animals; after successful mating.
- Maternal animals: All surviving animals; on day 21 post-partum; for non-pregnant females on or after day 25 post-coitum.
- The off-spring was sacrificed on day 21 after birth.

GROSS NECROPSY
- Gross necropsy consisted of full external and internal examinations of parental animals.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The following tissues of parental animals were prepared for microscopic examination and weighed, respectively: cervix, coagulating gland, epididymides, ovaries, pituitary gland, prostate, seminal vesicles, testes, uterus, vagina.

Statistics:

Linear regression analysis, followed by ANOVA incorporating Levene's test for homogeneity of variance was used for data on organ weights, weekly body weight, litter weights, offspring body weights. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances, the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney "U" test.
The non-parametric methods were also used to analyse implantation loss, offspring sex ratio, litter size and landmark developmental markers.
Chi-squared analysis was used for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
Kruskal-Wallis one-way non-parametric analysis of variance was used for the comparison of severity grades for the more frequently observed graded conditions.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Episodes of hunched posture, pilo-erection and tiptoe gait were evident in 500 mg/kg bw/day females during the final week of gestation.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male treated with 500 mg/kg bw/day was killed in extremis on day 93. One female from this treatment group was found dead on day 97 and a further two females were killed in extremis on days 99 and 100 following difficulties during parturition.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gain for parental males of 500 mg/kg bw/day group was generally lower (average -22 %, range -9 % to -43 %) than control animals throughout much of the treatment period, with statistical differences observed in Weeks 4, 5, 6 and 10.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Parental females treated with 500 mg/kg bw/day showed a notable reduction in food consumption (average -21 %, range -17 % to -28 %) throughout lactation, with statistically significant differences throughout 3 weeks.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The adult male treated with 500 mg/kg bw/day that was killed in extremis showed gaseous distension in the gastro-intestinal tract. The female treated with 500 mg/kg bw/day that was found dead around parturition had 21 foetuses in-utero. The two females from this treatment group that were killed in extremis both had dead/inactive fetuses in-utero and red/brown staining around the anogenital region and dark contents in the stomach or enlarged adrenals and an absent rougae on the non-glandular region of the stomach.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related microscopic changes were observed in the parental animals.

Effect levels

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on gestation length increase.

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In the one-generation study with rats, the NOAEL for parental toxicity was set at 25 mg/kg bw/day, based on gestation length increase in 100 and 500 mg/kg bw.

Executive summary:

Oral repeated dose toxicity of the test substance was studied in an OECD TG 415 study in compliance GLP using Sprague-Dawley rats. The substance was administered at dose levels of 0, 25, 100 and 500 mg/kg bw/day as a solution in arachis oil to groups of 24 rats/sex/dose for 76 days pre-mating and during maximum 21 days mating, after which males were killed, while females were killed and examined on day 21 post-partum. Females were killed and examined after day 25 post-coitum. One male treated with 500 mg/kg bw/day was killed in extremis on day 93. One female from this treatment group was found dead on day 97 and a further two females were killed in extremis on days 99 and 100 following difficulties during parturition. Episodes of hunched posture, pilo-erection and tiptoe gait were evident in 500 mg/kg bw/day females during the final week of gestation. Body weight gain for males of the 500 mg/kg bw/day group was generally lower (average -22 %, range -9% to -43 %) than control animals throughout much of the treatment period, with statistical differences observed in weeks 4, 5, 6 and 10. Females treated with 500 mg/kg bw/day showed a notable reduction in food consumption (average -21 %, range -17 % to -28 %) throughout lactation, with statistically significant differences throughout 3 weeks. There were no toxicologically significant effects on the concentration,motility or morphology of samples of epididymal sperm. There were no treatment-related effects on the concentration of homogenisation resistant epididymal or testicular spermatid counts. An increased gestation length was observed in the 100 and 500 mg/kg bw dose groups.At necropsy, the adult male treated with 500 mg/kg bw/day that was killed in extremis showed gaseous distension in the gastrointestinal tract. The female treated with 500 mg/kg bw/day that was found dead around parturitionhad 21 foetuses in-utero. The two females from this treatment group that were killed in extremis both had dead/inactive foetuses in-utero and red/brown staining around the ano-genital region and dark contents in the stomach or enlarged adrenals and an absent rougae on the non-glandular region of the stomach. No treatment-related microscopic changes were observed in the parental animals.Based on the observed increased gestation length, the NOAEL for parental toxicity was set at 25 mg/kg bw/day.