Reaction mass of Rosin, hydrogenated and [1R-(1α,4aβ,10aα)]-1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanthren-1-carboxylic acid

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

21 March 2002 to ....... 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline- and GLP compliant; no method of test substance analysis provided in the report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): Rosin
- Physical state: amber solid
- Analytical purity: unknown
- Impurities (identity and concentrations): unknown
- Composition of test material, percentage of components: complex mixture of destillation products
- Storage condition of test material: in the dark, at room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Sprague-Dawley / IGS (Crl:CG(SD) IGS BR of the Charles River Outbred strain (outbred albino)
- Source: Charles River (UK) Limited, Margate, Kent, England
- Age at study initiation: 6 wks
- Weight at study initiation: Males: 163-178 g; Females: 124-141 g
- Fasting period before study: none
- Housing:
-- 2 per cage, initially; poyplropylene cages, ca 42x27x20 cm, with stainless steel grid bottoms and mesh tops, stainless steel food hopper, polypropylene or polycarbonate water bottles (1/cage); male and female cages racked separately;
-- A few days prior to pairing for mating, males were transferred to individual grid-bottomed cages (ca 59x38.5x20 cm) of similar design. Mated females were transferred to individual 42x27x20 cm solid bottomed cages. Sterilised white wood shavings were provided as bedding and white paper tissue as nesting material where appropriate.
- Diet (e.g. ad libitum): Rat and Mouse Breeder Diet No. 3 (Expanded Ground) SQC, supplied by Special Diets Services Limited, Stepfield, Witham, Essex, UK was available to the animals ad libitum. A certificate of analysis is available in the report.
- Water (e.g. ad libitum): The animals had access to domestic mains water ad libitum via their water bottles. The supply is analysed regularly for dissolved and suspended materials, including a range of significant contaminants. A certificate of analysis is available in the report.
--- None of the contaminants revealed by the analysis of diet or water were considered to be present in high enough quantities to have affected the
outcome of the study.
- Acclimation period: The animals were acclimatised in the Inveresk animal room for 12 days prior to commencement of treatment. All the animals were clinically examined on arrival for signs of abnormality or disease. No such signs were found and the animals were accepted for use on the study.
- Animal identification: Each animal received a unique earmark identifying it individually within the study and corresponding to that animal’s number. Each animal was ascribed a cage card which was colour coded for treatment group, and was marked with the project number, group and animal numbers, sex and relevant treatment.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C ± 2°C
- Humidity (%): 50% ± 15%
- Air changes (per hr): 15 (minimum)
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 3 Apr 2002 To: 27 May 2002

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
not appropriate

DIET PREPARATION
Batches of diet were prepared weekly during the study, and used within 9 days of preparation.
An appropriate quantity of the test item was dissolved in a suitable volume of ethanol. This solution was added to a suitable quantity of untreated diet, then mixed for ca one hour with fan assisted venting to remove the ethanol to form a dose premix. A control premix was prepared using the same proportion of ethanol and untreated diet.
The diets for the Intermediate and High dose groups were prepared by dilution of the dose premix with untreated diet to give the desired concentrations. The Low dose diet was prepared by dilution of the High dose diet with untreated diet. The diet premixes were then placed on a Winkworth mixer for ca 20 min.
The Control diet was prepared by dilution of the control premix with untreated diet such that the diet contained the same proportion of premix as the High dose diet.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: maximum of 7 consecutive nights
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged individually (42x27x20 cm solid bottom cages, see also above)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diet formulations were analysed on 2 occasions during the study treatment period. Analysis of formulated diets was undertaken with regard to
concentration and homogeneity. On each occasion, triplicate samples were withdrawn from each formulated diet containing test item, and from the Control diet. The samples were analysed by the Toxicology Support Laboratory, using a method supplied by the Sponsor and previously validated in the Inveresk laboratory under a separate protocol and contract (Inveresk Project No. 419841).

Duration of treatment / exposure:

The males were treated for at least 4 weeks overall, starting from 2 weeks prior to mating until termination.
The females were treated until termination, commencing 2 weeks prior to mating until at least Day 4 of lactation.

Frequency of treatment:

The animals were dosed continuously via the diet. The concentration of the test item in the diet remained constant throughout the treatment period.
Control animals received control diet.

Details on study schedule:

- Age at mating of the mated animals in the study: ca. 10 weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1000. 3000. 10000
Basis:
nominal in diet
ppm

Doses / concentrations 2

Remarks:

Doses / Concentrations:
962, 2760, 9862
Basis:
analytical conc.
ppm

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected and agreed with the Sponsor, following evaluation of existing toxicological data. This included data from a one week dose range finding study in rats carried out under a separate contract and project number at Inveresk (Inveresk Project No. 493160).
- Rationale for animal assignment (if not random): On arrival from the suppliers, the animals were housed in cages which had been allocated to specific treatment groups using a series of randomly sequenced numbers. Cages in any one treatment group were evenly distributed through the caging system so each full rack contained representatives from all treatment groups.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS (Viability): Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: nce daily. The nature, onset, duration and intensity of any signs were recorded.


BODY WEIGHT: Yes
- Time schedule for examinations: Male weights were recorded once during the week prior to the commencement of dosing and once weekly thereafter until termination.
Female weights were recorded once during the week prior to the commencement of dosing, and weekly thereafter until the start of the mating period, and then on Day 0 of gestation (the day of detection of a positive mating sign) followed by Days 7, 14 and 20 of gestation, and then Days 1 and 4 of lactation (where Day 0 = the day of parturition).

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OTHER:
CAGE SIDE OBSERVATIONS (Viability): Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: Once daily. The nature, onset, duration and intensity of any signs were recorded.
- Male weights were recorded once during the week prior to the commencement of dosing and once weekly thereafter until termination.
Female weights were recorded once during the week prior to the commencement of dosing, and weekly thereafter until the start of the mating period, and then on Day 0 of gestation (the day of detection of a positive mating sign) followed by Days 7, 14 and 20 of gestation, and then Days 1 and 4 of lactation (where Day 0 = the day of parturition).

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Food consumption for each animal determined: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- For all animals, the weight of food consumed by each cage was recorded once weekly commencing during the week prior to the commencement of
dosing, until pairing for mating. All animals were fed ad libitum during mating, but following completion of mating weekly consumption was recommenced for males, until termination.
For mated females, the amount of food consumed was recorded over Days 0-7, 7-14 and 14-20 of gestation, and Days 0-4 of lactation.

OTHER:
- Observations on Females with Litters During Lactation:
The females were allowed to litter normally. The day on which parturition commenced was designated Day 0 of lactation. The duration of gestation was evaluated.

Oestrous cyclicity (parental animals):

not determined

Sperm parameters (parental animals):

not appropriate

Litter observations:

The number of live pups born and the number found dead in each litter were recorded as soon as possible after completion of parturition. The live pups were sexed, counted, examined for the presence of milk in the stomach and for any externally visible abnormalities daily until Day 4 of lactation. Data recorded after Day 4 of lactation was not reported, but were retained in the study data.
Any deficiencies in maternal care were recorded. These included, but were not limited to, inadequate construction and cleaning of the nest, pups left
scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding. Detailed information is retained in the raw data.
The live pups were weighed en masse (by sex) on Days 1 and 4 of lactation.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: The males were sacrificed once mating was completed, and the animals had been dosed for at least 4 weeks.
- Maternal animals: The females were sacrificed with their litters between Day 4 and Day 7 of lactation.

GROSS NECROPSY
- A random order was used for the necropsy of the adult animals.
- Gross necropsy: The cranial, thoracic and abdominal contents were examined macroscopically; any findings were recorded and representatives samples of abnormal tissues were taken and fixed in neutral buffered 10% formalin, as appropriate.
The female reproductive tract was dissected out and the number of implantation sites in the uterus was recorded.
The adult carcasses were discarded after tissue sampling.

HISTOPATHOLOGY / ORGAN WEIGHTS
For the males the testes and epididymides were weighed, and the epididymides, seminal vesicles, coagulating gland, prostate gland and pituitary were fixed in 10% neutral buffered formalin. The testes were fixed in Bouin’s fluid.
Females: The reproductive tract was fixed in 10% formalin.
Histological examination was conducted on Control and High dose animals only. A section from each ovary and each epididymis, and a transverse
section from each testis were stained with Haematoxylin and Eosin (H&E) and a further section from each testis was stained with PAS-Haematoxylin. These sections were evaluated by a pathologist. No histological examination was conducted on the other preserved tissues and organs.

Postmortem examinations (offspring):

SACRIFICE
The pups were sacrificed at the same time as their mother (see above).

GROSS NECROPSY
The pups were examined for externally visible abnormalities. The pups were discarded following examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
No histopathology was performed.

Statistics:

Body weight and food consumption data in animals prior to mating were subjected to analysis of variance or the Kruskal-Wallis non-parametric analysis as appropriate.
Organ weight data were analysed by analysis of variance and analysis of covariance using the terminal bodyweight as the single covariate.
Histology data were analysed by Fisher’s Exact Probability test.

Reproductive indices:

The following indices of fertility were evaluated for each group:
Fertility Index (female) = Number Pregnant / Number Paired
Fertility Index (male) = Number Siring a Litter / Number Paired
Gestation Index = Number bearing live pups / Number pregnant
Birth Index = Total number of pups born (live and dead) / Number of implantation scars

Offspring viability indices:

Live Birth Index = Number of pups live on Day 0 of lactation / Total number born (live and dead)
Viability Index = Number of pups live on Day 4 of lactation / Number live on Day 0

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

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:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Animal 65 (3000 p.p.m.) was found dead after having given birth to 7 live pups. Examination of the dam revealed a further 8 pups in utero. No abnormalities were detected following macroscopic examination of the dam or the pups, and the cause of death was not established.
The clinical observations and necropsy findings for the other animals were considered to be consistent with those normally seen in rats of this age and strain.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Treatment at 10000 p.p.m. was associated with a decrease in weight gain or weight loss over the first few weeks of treatment. In males decreased weight gain was evident over a 2 week period, whilst in females the findings were more severe, with weight loss, over a period of one week. After the 2 weeks for males, and one week for females, mean weight gain was similar to the Control value, although the earlier deficit in absolute body weight still
remained. For pregnant females weight gain was slightly decreased during the first half of gestation.
There was a slight decrease in body weight gain over Week 0 -Week 4 in males treated at 3000 p.p.m, when compared to Controls. A marginal decrease in weight gain in males at 1000 p.p.m. was noted over the first week of treatment, but owing to the minor nature of the change, these findings could
not be positively attributed to treatment. The body weight gains for females at 1000 p.p.m. and 3000 p.p.m. were similar to Control.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
At 10000 p.p.m, the achieved intake in the first week of treatment for bothsexes was lower than in the second week. Normally a higher intake would be expected in the first week, compared with the second. Also, there was a decreased intake in the first week of gestation at this level. During these weeks, the achieved intakes at this level were slightly less than proportional to the diet concentrations.
At other times, the achieved intake was essentially proportional to the diet concentrations.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no effects of treatment on mating performance, fertility or the duration of gestation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Testes and epididymides weight were essentially similar in all groups.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no effects of treatement noted in any test substance group.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no histology findings that could be attributed to treatment with
Rosin.

LITTER SIZE
At 10000 p.p.m. there was a slight decrease in the mean number of implant
sites per pregnancy and a subsequent slight reduction in litter size.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

no mortality observed

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:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
At 10000 p.p.m. there was a slight decrease in the mean number of implant sites per pregnancy and a subsequent slight reduction in litter size. Litter survival, as indicated by the birth index and viability index, was similar in all groups.

BODY WEIGHT (OFFSPRING)
At 10000 p.p.m. mean litter and pup weights were slightly reduced compared to the Controls

ABNORMALITIES AMONG PUPS
There were no obvious external abnormalities noted in the pups at any of the dose levels applied.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

3 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The no effect level for adults was considered to be 1000 p.p.m. (corresponding to doses between 71 and 109 mg/kg bw), and there were no effects detected on reproduction/development at levels of 1000 (corresponding to doses between 71 and 109 mg/kg bw) and 3000 p.p.m. (corresponding to doses between 208 and 352 mg/kg bw) as assessed by this study.

Executive summary:

Treatment with Rosin at 10000 p.p.m. (corresponding to doses between 704 and 1025 mg/kg bw) was associated with reduced weight gain/ weight loss and reduced food consumption for the first few weeks of treatment. The deficits in body weight were never regained. Food consumption was reduced throughout gestation and body weight gain was reduced during the first half of gestation.

At 10000 p.p.m. the mean number of implant sites per pregnancy was slightly decreased resulting in a subsequent reduction in litter size. Mean litter and pup weights were also slightly reduced. The effects on implantation, litter size and foetal weight may be secondary to the effects on food intake and subsequent weight gain in the adult females.

Body weight gain was slightly reduced in males at 3000 p.p.m. (corresponding to doses between 208 and 352 mg/kg bw). Any minor change in the food consumption at this level was too small to be attributed to treatment.

The no effect level for adults was considered to be 1000 p.p.m. (corresponding to doses between 71 and 109 mg/kg bw), and there were no effects detected on reproduction/development at levels of 1000 p.p.m. and 3000 p.p.m. as assessed by this study.