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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to guideline protocol

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Rosin, fumarated
- Molecular formula (if other than submission substance):
- Molecular weight (if other than submission substance):
- Smiles notation (if other than submission substance):
- InChl (if other than submission substance):
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type: UVCB, distillation products
- Physical state: amber solid
- Analytical purity: normal commercial sample
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Isomers composition:
- Purity test date:
- Lot/batch No.: TWR01075-15FTOR-9.19.01
- Expiration date of the lot/batch: 21 September 2006
- Radiochemical purity (if radiolabelling):
- Specific activity (if radiolabelling):
- Locations of the label (if radiolabelling):
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions:
- Storage condition of test material: in the dark at room temperature.
- Other:

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England
- Age at study initiation: (P) 6 x wks on arrival;
- Weight at study initiation: on arrival (P) Males: 140-169 g; Females: 111-138 g;
- Fasting period before study: none
- Housing: 2 per cage initially, in polypropylene cages, with stainless steel grid bottoms and mesh tops. A few days prior to pairing for mating, males were transferred to individual cages of similar design. Mated females were transferred to individual solid bottomed cages.
- Use of restrainers for preventing ingestion (if dermal): n/a
- Diet (e.g. ad libitum): Ad libitum. Rat and Mouse Breeder Diet No. 3 (Expanded Ground) SQC, (Special Diets Services Ltd., Essex, UK)
- Water (e.g. ad libitum): Ad libitum, domestic mains water.
- Acclimation period: 13 days

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

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: diet
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly and used within 15 days of preparation
- Mixing appropriate amounts with (Type of food): Rat and Mouse Breeder Diet No. 3 (Expanded Ground) SQC
- Storage temperature of food: No data.
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.
Diet prepared for Week 1 and Week 4 of treatment was sampled. 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 laboratory.
Details on mating procedure:
- M/F ratio per cage: 1
- Length of cohabitation: Maximum 7 consecutive nights.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility (following a rest period of 2 nights).
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged (how): Individually in solid bottomed cages with white wood shavings as bedding and white paper tissue as nesting material if appropriate
- Any other deviations from standard protocol:no
Duration of treatment / exposure:
Males: at least 4 weeks overall, starting from 2 weeks prior to mating until termination.
Females: commencing 2 weeks prior to mating, then through mating until termination after Day 4 of lactation.
Frequency of treatment:
Continuous
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1000, 3000, 10000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels selected and agreed with 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 the laboratory.

There were no replacement of study animals.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily for viability early in the morning and again as late as possible on each day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily. Nature, onset, duration and intensity of any signs were recorded

BODY WEIGHT: Yes
- Time schedule for examinations: Males - once during the week prior to the commencement of dosing and once weekly thereafter until termination.
Females - once during the week prior to the commencement of dosing, and weekly thereafter until the start of the mating period. Then on Day 0 of gestation followed by Days 7, 14, and 20 of gestation, and then Days 1 and 4 of lactation (where Day 0 of lactation is day of parturition).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OTHER: LABORATORY INVESTIGATIONS: Samples were initially obtained from 5 males and 5 females from each dose group. For males, the first 5 animals in each group were tested. For females, where possible, the first 5 animals to have reared their litter to Day 6 of lactation were tested. One additional sample was taken for female controls to ensure a total of 5 samples. Additional samples were taken from 2 high dose females on day 7 of lactation owing to problems with obtaining sufficient samples for analysis.
Haematology: Haemoglobin, RBC count, Haematocrit, WBC count, MCV, mean cell haemoglobin, mean cell haemoglobin concentration, platelets, differential WBC count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unclassified cells.
Coagulation: Prothrombin time
Clinical chemistry: urea, glucose, aspartate aminotransferase, alanine aminotransferase, sodium potassium, chloride, total protein, albumin, A:G ration, creatinine, calcium, phosphate, total bilirubin, cholesterol, alkaline phosphatase.

Postmortem examinations (Parental animals)
SACRIFICE
- Maternal animals: All surviving animals after Day 4 of lactation

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively. Histological examination was conducted on control and high dose animals only.
Ovaries and uterine content:
Estrous cyclicity was not measured.
Fetal examinations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, other: any deficiencies in maternal care were recorded.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (Offspring)
Pups were examined for externally visible abnormalities and discarded following examination


Statistics:
Body weight, food consumption (prior to mating for females), haematology and clinical chemistry were statistically analysed for homogeneity of variance using the 'F-max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons made via Student's t-test using Fisher's F-protected LSD. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogenous then Kruskal-Wallis ANOVA was used.
Organ weights were analysed as above and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate.
Histology incidence data were analysed using Fisher's Exact Probability Test.
All tests were two-sided and performed at the 5% significance level.

Indices:
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
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: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Decrease in mean body weight at 3000 and 10,000 ppm, an increase in total bilirubin at 10,000 ppm, and a decrease in adrenal gland weight at 10000 ppm

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
3 000 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
10 000 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:not examined

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
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, rosin, fumarated is not a reproductive toxicant and it is not selectively toxic to the developing fetus. Rosin, fumarated 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:

Rosin, fumarated was administered in the diet to female rats at concentrations of 0, 1000 ppm (79-108 mg/kg bw/d), 3000 ppm (196-292 mg/kg bw/d), and 10,000 ppm (449-995 mg/kg bw/d) throughout pregnancy until termination on Day 4 of lactation. Food consumption and mean body weights were decreased at 10,000 ppm and 3000 ppm, with high dose animals also showing an increase in total bilirubin and decreased adrenal weight.  The lower of these two values will be used as the maternal (systemic) NOAEL. This is considered scientifically defensible since, apart from poor palatability and associated body weight reduction following exposure to 10000 ppm test substance, no clearly adverse effects were apparent. With regard to litter parameters, there was a slight decrease in the mean number of implant sites per pregnancy and a consequent slight reduction in litter size at birth in the high dose group. A slight reduction in litter size between Day1-4 of lactation at 3000 ppm was due to the loss of most pups in one litter. As there were no effects of treatment on litter survival at 10,000 ppm the findings at 3000 ppm are considered to be incidental. Based on these results, the NOAEL for developmental effects was considered to be 10,000 ppm (449-995 mg/kg bw/d).