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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 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
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
not applicable
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
Sex:
male/female
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

IN-LIFE DATES: From: March 31, 2003 To: June 26, 2003

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.
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
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.
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
Details on study schedule:
The females were allowed to litter normally. The day on which parturition commenced was designated Day 0 of lactation.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1000, 3000, 10000ppm
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.
Positive control:
none

Examinations

Parental animals: Observations and 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.
Oestrous cyclicity (parental animals):
No.
Sperm parameters (parental animals):
Parameters examined in male parental generation:
testis weight, epididymis weight, seminal vesicle examination, prostate weight
Litter observations:
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 (parental animals):
SACRIFICE
- Male animals: All surviving animals following 4 weeks of treatment
- 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.
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.
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: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
10000ppm
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
10000 and 3000ppm
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
10000 and 3000ppm
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: 10000 and 3000ppm

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
10000ppm

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Abnormal coloured urine in 2/10, 1/10 and 3/10 animals per sex at 1000, 3000 and 10000ppm, respectively. At 10000ppm it was generally observed from Day 7 onwards and lasted no longer than 3 days. At lower levels it did not occur until Day 15 or later, and was present for one day only.
At 10000ppm, soft faecal output noted in 4/10 males (over Days 10-11 treatment) and 6/10 females (on Day 10).
At 3000ppm soft faecal output evident in 2/10 males.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Treatment at 3000 and 10000ppm associated with decrease in mean body weight gain and food consumption in both sexes.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
At 10000ppm both sexes, and among males at 3000ppm, the achieved intake in the first week of treament was lower than in the second week. Among females at 10000ppm there was a decreased intake over Days 0-7 and 14-20 of gestation and during lactation.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
At 10000ppm there was a slight increase in the number of nights to a positive mating sign and in the duration of gestation. The fertility indices were lower than those of the control group but were considered to be within normal ranges. At 3000ppm all animals mated within 4 nights; the slight increase in the median number of nights to positive mating sign was therefore considered to be coincidental.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In males at 10000ppm there was a slight decrease in absolute lung and spleen weights (statistically significant) but following caviance analysis there differences from control were no longer evident.
In females mean kidney, spleen, adrenal gland, ovary and uterus weights were significantly reduced at 10000ppm. Following covariance analysis a decrease respect to controls was only evident for adrenal weight.

GROSS PATHOLOGY (PARENTAL ANIMALS)
None remarkable

HISTOPATHOLOGY (PARENTAL ANIMALS)
None remarkable. Increase in thymic atrophy in females at 10000ppm conseidered non treatment related.

OTHER FINDINGS (PARENTAL ANIMALS)
Haematology: At 10000ppm in males only there were slight decreases in haemoglobin, red blood cell count and haematocrit. At 3000ppm in males a statistical decrease in monocytes was considered incidental as was not associated with dose.

Clinical Chemistry: Total bilirubin was significantly increased in both sexes at 10000ppm

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
3 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Parental NOAEL, equivalent to 221-288 mg/kg bw/d for males, and 196-292 mg/kg bw/d for females
Dose descriptor:
NOAEL
Effect level:
10 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive NOAEL, equivalent to 651-889 mg/kg bw/d for males, and 449-995 mg/kg bw/d for females

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
10000ppm
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
10000ppm
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 10000ppm there was a slight decrease in the mean number of implant sites per preganancy and a consequent slight reduction in litter size at birth. The slight reduction in litter size between Day1-4 of lactation at 3000ppm reflects the loss of most pups in one litter. As there were no effects of treatment on litter survival at 10000ppm the findings at this level are considered to be incidental.

BODY WEIGHT (OFFSPRING)
At 10000ppm mean litter weight was slightly reduced compared to the controls, reflecting the decrease in litter size. Pup weights were marginally lower than those of the controls on Day 4 of lactation.

GROSS PATHOLOGY (OFFSPRING)
One control pup had a lesion on ventral abdomen

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

none

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 rats at concentrations of 0, 1000 ppm (males 72-89 mg/kg bw/d; females 79-108 mg/kg bw/d), 3000 ppm (males 221-288 mg/kg bw/d; females 196-292 mg/kg bw/d), and 10,000 ppm (males 651-889 mg/kg bw/d; females 449-995 mg/kg bw/d). The males were treated for 2 weeks prior to mating, through until necropsy after 4 weeks of treatment. The females were treated for 2 weeks prior to mating, then through mating, gestation and until termination on at least Day 4 of lactation. Food consumption and mean body weights were decreased in parental animals of both sexes at 10,000 ppm and 3000 ppm, with high dose animals also showing an increase in total bilirubin (both sexes) and decreased adrenal weight (females only).  The lower of these two values will be used as the parental (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 reproductive 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 reproductive parameters was considered to be 10,000 ppm (males 651-889 mg/kg bw/d; females 449-995 mg/kg bw/d).