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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 421 and GLPs.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
-Test substance (as cited in report): Rosin
-Physical appearance: Amber colored solid
-Storage: In the dark at room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Test animals:
-Strain: Crl: CD®(SD) IGS Br strain (outbred albino)
-Source: Charles River (UK) Ltd., Margate, Kent, UK
-Sex: Male and female
-Age at study initiation: approximately 6 weeks
-Acclimation period: 12 days
-Weight at study receipt: Males 163-178g
Females 124-141g
-Housing: Animals were initially housed 2 per cage in polypropylene cages suspended over tray-lined absorbent paper for collection of waste. Male and female cages were housed and racked separately except during mating. Prior to mating, males were transferred to individual grid-bottomed cages. After mating, females were transferred to individual solid-bottomed cages. Sterilized white wood shavings were provided as bedding and white paper tissue as nesting material.
-Diet: Rat and Mouse Breeder Diet No. 3 (Expanded Ground) SQC (Special Diets Services Ltd., Stepfield, Witham, Essex, UK) ad libitum
-Enrichment: Wooden chewsticks
-Water: local municipality, ad libitum
-Method of animal identification: unique earmark
-Method of animal distribution: The animals were housed in cages which had been allocated to treatment groups using a series of randomly sequenced numbers. Cages in any one treatment group were evenly distributed throughout the caging system so each rack contained representatives from all treatment groups.

Environmental Conditions:
-Temperature (°C): 20 ± 2
-Humidity (%): 50 ± 15
-Photoperiod: 12 hours light/dark
-Air exchanges: minimum 15 air exchanges per hour

Administration / exposure

Route of administration:
oral: feed
Vehicle:
ethanol
Details on exposure:
Test formulations:
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 ethanol and added to the untreated diet, then mixed for approximately one hour with fan assisted venting to aid removal of the ethanol. A control premix was prepared using the same proportion of ethanol and untreated diet. The high- and mid-dose diets for the study 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 placed on a Winkworth mixer for approximately 20 minutes.

Exposure:
The study consisted of four phases: pre-mating (14 days), mating (up to 7 days), gestation (21-23 days) and early lactation (4 days) for a total of 41-45 exposure days for females. Males were treated for at least 4 weeks overall (2 weeks prior to mating and two weeks during and subsequent to mating). The rats were offered feed containing the test substance at 0, 1000, 3000, or 10000 ppm ad libitum until the rats were euthanized. All surviving females that delivered a litter were euthanized and necropsied on Day 4 postpartum. Pups were exposed only in utero or during lactation. All surviving pups were euthanized on Day 4 postpartum.     
Details on mating procedure:
Pairing was 1 male with 1 female within the same treatment group. Each female was transferred to the cage of an appropriate male and remained there until mating was confirmed either from a sperm positive vaginal smear or a copulatory plug. The day of observation of a copulatory plug in situ and/or sperm in the lavage was designated as Gestation Day 0.

Each female was left with the male for a maximum of 7 nights. If confirmation of mating was not detected during that time, the female was given a rest period of 2 nights, and then placed with a second male from the same group for a maximum of 7 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Diet formulations were analysed twice during the study for concentration and homogeneity. On each occasion, triplicate samples were taken from each test diet and from the control diet.
The analysed concentrations from both sample timepoints were found to be within ± 10% of the nominal concentration. Homogeneity was confirmed by the low coefficient of variation (2.9% or less).
Duration of treatment / exposure:
Females: 41-45 days
Males: 30 days
Frequency of treatment:
Daily
Details on study schedule:
In-Life Study Dates:
-Study Initiation Date: March 21, 2002
-Experimental Start Date: April 9, 2002
-Experimental Completion Date: July 18, 2002
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1000 ppm, 3000 ppm and 10,000 ppm
Basis:
nominal in diet
Equivalent to: 0, 84, 248 and 786 mg/kg bw/d for males (weeks 1-4); 0, 102, 309 and 869 mg/kg bw/d for females (to PND4)
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
Dose levels were selected in consultation with the Sponsor after evaluation of existing toxicological data.

Doses Received:
Mean daily doses of the active ingredient received were:
-Males:
Four week average:
1000 ppm: 84.0 mg/kg bw/day
3000 ppm: 248.0 mg/kg bw/day
10000 ppm: 786.3 mg/kg bw/day

-Females:
Pre-mating:
1000 ppm: 98.0 mg/kg bw/day
3000 ppm: 287.0 mg/kg bw/day
10000 ppm: 791.0 mg/kg bw/day

Gestation:
1000 ppm: 94.3 mg/kg bw/day
3000 ppm: 266.7 mg/kg bw/day
10000 ppm: 826.0 mg/kg bw/day

Lactation:
1000 ppm: 109.0 mg/kg bw/day
3000 ppm: 352.0 mg/kg bw/day
10000 ppm: 1025.0 mg/kg bw/day

Examinations

Parental animals: Observations and examinations:
Clinical Observations:
All animals were examined for toxicity on a daily basis and any reaction, its onset, duration and intensity were recorded. Animals were examined twice a day for mortality and moribundity.

Body Weights:
Body weights for males were recorded once during the week prior to the start of the study and weekly until euthanasia. Females weights were recorded once during the week prior to the start of the study, weekly until the commencement of the mating period, on Gestation Days 0, 7, 14, and 20, and on Lactation Days 1 and 4.

Food Consumption:
For all animals, the weight of food consumed by each cage was recorded once a week starting during the week prior to the start of the study until mating when the animals were fed ad libitum. Females had food consumption values recorded on Gestation Days 0, 7, 14, and 20, and on Lactation Days 1 and 4. Feed consumption measurements for males were recommenced weekly following completion of mating.
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
Clinical Observations:
The number of live pups born, the sex, and the number found dead in each litter were recorded after completion of parturition. All pups were observed once daily for clinical signs, externally visible abnormalities, presence of milk in the stomach, moribundity, and mortality.

Body Weights:
Body weights for live pups were measured as a group by sex on Postnatal Days 1 and 4.

Maternal Care: Any deficiencies in maternal care were recorded. These included inadequate construction and cleaning of the nest, pups left scattered and cold, physical abuse of the pups, or inadequate lactation or feeding.
Postmortem examinations (parental animals):
Adult animals were sacrificed by carbon dioxide asphyxiation, weighed then exsanguinated by severance of a major blood vessel. The adult animals were subjected to a gross necropsy, in which the cranial, thoracic and abdominal contents were examined macroscopically and any findings were recorded and gross lesions were preserved in neutral buffered 10% formalin. Additional tissues retained for possible examination included: testes (fixed in Bouin's solution), epididymides, seminal vesicles, coagulating gland, prostate gland, female reproductive tract and pituitary gland were fixed in 10% neutral buffered formalin.

Organ weights:
For males, the epididymides and testes were weighed; paired organs were weighed together.

Histopathology:
Histopathological examination was conducted on animals of the control and the 10000 ppm group. A section from each ovary and epididymis and a transverse section from each testis were stained with Haematoxylin and Eosin (H&E). A further section from each testis was stained with PAS-Haematoxylin and all sections were evaluated by a pathologist. No histological examination was conducted on the other preserved tissues and organs. Adult carcasses were discarded after tissue sampling.
Postmortem examinations (offspring):
The pups were euthanized by injection of sodium pentobarbitone, examined for externally visible abnormalities and discarded.
Statistics:
Body weight and food consumption data were subjected to analysis of variance or Kruskal-Wallis non-parametric analysis. Organ weights were analysed by analysis of variance and analysis of covariance using the terminal body weight as the single covariate. Histology data were analysed by Fisher's Exact Probability test. All statistical tests were two-sided and statistical significance was ascribed at the 5% significance level.
Reproductive indices:
Fertility Index (female) = Number Pregnant ÷ Number paired

Fertility Index (male) = Number siring a litter ÷ Number paired

Gestation Index = Number bearing live pups ÷ Number pregnant
Offspring viability indices:
Birth Index = Total number of pups born (live and dead) ÷ Number of implantation scars

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 animals)

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
Gross pathological findings:
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:
effects observed, treatment-related

Details on results (P0)

Clinical observations and mortality:
One dam in the 3000 ppm group was found dead after giving birth to 7 pups and gross necropsy revealed an eighth pup in utero. No abnormalities in the dam or pups were detected. There were no clinical observations in other animals that could be attributed to treatment with the test substance.

Body weights:
Treatment of the 10000 ppm group was associated with a decrease in weight gain or weight loss over the first few weeks of treatment. Decreased weight gain was evident, in males, over a 2 week period, while in females the findings were more severe, with weight loss during the first week on study. After this 2 week (males) and 1 week (females) period, mean weight gain was similar to controls. Pregnant female weight gain was slightly decreased during the first half of gestation. A slight decrease in body weight gain from study start to Week 4 in males was observed at 3000 ppm compared to controls and a marginal decrease in weight gain in males at 1000 ppm was noted over the first week of treatment, but these findings could not be attributed to test substance treatment. No weight changes were noted in females at the two lower dose levels.

Food consumption:
Food consumption was significantly reduced in the 10000 ppm animals for the first 2 weeks of treatment, for both sexes. Food consumption was also slightly reduced throughout gestation and lactation. Consumption at the two lower levels was similar to that of controls.

Test substance intake:
At 10000 ppm, intake in the first week of treatment was lower than the second week for both sexes. There was also a decreased intake in the first week of gestation at this dietary level. During these weeks, achieved intakes at this level were slightly less than proportional to the diet concentrations.

Reproductive performance:
There were no effects of treatment on mating performance, fertility or the duration of gestation. A slight decrease in the mean number of implantation sites per pregnancy and a subsequent slight reduction in litter size were observed in dams exposed to 10000 ppm of the test substance. Litter survival, as indicated by the birth index, live birth index, and viability index, was similar in all groups.

Organ weights:
No effects

Gross pathology:
No effects

Histopathology:
There were no histology findings that could be attributed to the test substance.

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
Reproductive Toxicity
Effect level:
3 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The mean number of implantation sites per pregnancy was slightly decreased resulting in a subsequent slight reduction in litter size in dams exposed to 10000 ppm (NOAEL 3000 ppm, equivalent to 248-309 mg/kg bw/d).

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
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:
not specified
Histopathological findings:
not examined

Details on results (F1)

Viability:
No effects on mean number of live pups born compared to controls.

Clinical signs:
No effects, observations were observed to a similar extent in all groups.

Body weight:
Pups exposed in utero to 10000 ppm had slightly reduced body weights compared to controls. All other groups were similar to controls.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
Developmental Toxicity
Generation:
F1
Effect level:
3 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Mean litter and pup weights were slightly reduced compared to controls in dams exposed to 10000 ppm (NOAEL 3000 ppm, equivalent to 288 mg/kg bw/d on GD0-PND4).

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
There were no test material-related effects on mating performance, fertility or the length of gestation in a reproduction and developmental toxicity screening study in which adult male and female rats were exposed via the diet to nominal concentrations of 0, 1000, 3000 or 10000 Gum Rosin during premating, mating, gestation and early lactation for a total of 30 days in males and 41-45 days in females. At 10000 ppm, there were no effects of treatment on mating performance, fertility or the duration of gestation and no gross or microscopic effects on reproductive organs in either sex. The mean number of implant sites per pregnancy at 10000 ppm was slightly decreased resulting in a subsequent slight reduction in litter size. Mean litter and pup weights were also slightly reduced at 10000 ppm. There were no obvious external malformations noted in the pups at any of the dose levels in this study. The effects on implantation, litter size and fetal weight may be secondary to the effects on food intake and subsequent weight gain in the adult females. The no-observed-effect-level (NOEL) for reproductive and neonatal toxicity was considered to be 3000 ppm when rats were exposed to Gum Rosin at dose concentrations of 0, 1000, 3000, or 10000 ppm for 41-45 days (females) or 30 days (males) in the diet.

In a well-conducted reproductive and developmental toxicity screening study in rats, there was no clear evidence of test substance-related effects on reproduction of F0 males and females or on survival and development of F1 pups. Based on this information, Gum Rosin is not a reproductive toxicant and it is not selectively toxic to the developing fetus. Gum Rosin is not classified for “Developmental or Reproductive Toxicity” according to Directive 67/548/EEC, UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) or EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
Executive summary:

In a reproductive/developmental toxicity screening study, 10 rats/sex/group were exposed to Gum Rosin at dose concentrations of 0, 1000, 3000, or 10000 ppm for 41-45 days (females) or 30 days (males) in the diet. Treatment with Gum Rosin at 10000 ppm was associated with reduced weight gain/weight loss and reduced food consumption in the parental generation and a slight decrease in the mean number of implantation sites resulting in a subsequent slight reduction in litter size. Body weight gain reductions were also observed in males exposed to 3000 ppm Gum Rosin. Adverse effects in the F1 pups were limited to slightly reduced litter and pup weights. Based on the results of the present study, the NOAEL for reproductive/developmental toxicity in Sprague-Dawley rats was considered to be 3000 ppm for males (equivalent to 248 mg/kg bw/d) and females (equivalent to 309 mg/kg bw/d) and the NOAEL for subchronic toxicity was considered to be 1000 ppm in males (equivalent to 84 mg/kg bw/d) and 3000 ppm in females (equivalent to 309 mg/kg bw/d) based upon reduced feed consumption and lower weight gain in animals consuming higher dietary concentrations of the test material.