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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:
experimental study
Adequacy of study:
supporting study
Study period:
2007-08-02 to 2008-10-13
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Study summary available only. Original reference was not obtainable, but the study was approved by the OECD procedure on Mutual Acceptance of Data (MAD).
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Titanium dioxide (supplier: Sigma-Aldrich Co.)
- Physical state: white powder
- Analytical purity: 99.4%
- Lot/batch No.: 70230
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: ORIENTBIO INC., Korea
- Age at receipt (males and females): 7 weeks old
- Age at start of administration (males and females): 8 weeks old
- Weight at receipt: males: 209.6 to 225.8 g; females: 148.9 to 163.8 g
- Weight at start of administration: males: 281.5 to 324.1 g; females: 178.6 to 207.8 g
- Housing: stainless wire mesh cages, 280W x 500L x 200H (mm). Polycarbonate cage 240W x 390L x 175H (mm); two animals per cage (during pre-mating periods)
- Diet (ad libitum): pelleted feed for experimental animals (purchased from PMI Nutritional International, Inc. Indiana, USA), irradiated with gamma-ray
- Water (ad libitum)
- Acclimation period: approximately 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 3 °C
- Relative humidity: 50 % ± 10 %
- Air changes: 10 to 20 clean, fresh, filtered air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle, 8 AM to 8 PM via automated timer

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% methylcellulose (MC) solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Methylcellulose was dissolved in distilled water and 1% (w/w) solution was prepared. The test item of the treatment group was suspended in 1% methylcellulose solution.

VEHICLE - 1% methylcellulose (MC) solution
- Lot no.: 095K0189
- Storage condition: room temperature
- Supplier: Sigma-Aldrich Co. Ltd
Details on mating procedure:
- M/F ratio per cage: 1 female / 1 male
- Length of cohabitation: 2 weeks
- Females in which mating was not detected during the first mating period were re-mated with the males within the same group for 2 days.
- Proof of pregnancy: the first 24 hour-period following mating was designated as day 0 of pregnancy, if sperm or vaginal plug were detected.

Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
- Males: total of 40 days (2 weeks prior to mating, mating period and 2 weeks post mating period)
- Females: 2 weeks prior to mating date up to day 3 of lactation including the mating and gestation period
Frequency of treatment:
once a day
Doses / concentrations
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 males / 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
no data
Positive control:
no data

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day during the non-treatment period and every day before and after dosing during the treatment period, and were recorded using Path/Tox system.
- Cage side observations checked: clinical signs, intoxication and mortality; nursing behaviour of dams

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations:
Males and females were weighed once a week during the premating and mating periods. Measurement of body weights during the gestation and lactation periods was conducted as follow:
1) Pregnant animals: Days 0, 7, 14 and 20 of gestation
2) Lactation animals: Days 0 and 4 of lactation

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Rats were supplied with a certain amount of diet on days of body weight measurement and food consumption were recorded for the animals on the following days. On mating start day, a diet was supplied on the day before mating start and food consumption were recorded on the following day. In addition, on day 4 of lactation a diet was supplied on day 3 of lactation and food consumption was recorded on the following day.

WATER CONSUMPTION AND COMPOUND INTAKE: No data

OBSERVATION ON GESTATION AND PARTURITION: Yes
- successful copulation was decided by indentifying implantation sites in uterus at caesarean section.
- abortion, premature delivery and dystocia or prolonged parturition were observed.
- gestation length was observed
- pregnancy period was observed
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
The following organ weights of all adult animals were measured and relative organ weights were calculated based on the terminal body weight:
- testis
- epididymis
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in the offspring at parturition: litter size, stillbirths, sex of pups, body weight of pups, runts (pups that were significantly smaller than corresponding control pups), and external anomalies of live pups
The following parameters were examined in the offspring during lactation: viability and body weights of pups (Day 0 and 4 of lactation) were observed.d.


Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all males were sacrificed after the mating period and at least until the minimum total dosing period of 28 days had been completed.
- Maternal animals: females were terminated on Day 4 of lactation, while anesthethetized with CO2 gas.

GROSS NECROPSY
For all animals, gross necropsy consisted of a complete external and internal examination and identification of all abnormal findings.
At necropsy of females, corpora lutea and implantation sites were counted.

HISTOPATHOLOGY / ORGAN WEIGHTS
Following organs of all adult animals were fixed: testis, epididymis, seminal vesicle, prostate, ovary, vagina, cervix, and uterus
All organs except testis and epididymis were taken and fixed with 10% neutral buffered formalin solution and testis and epididymis were preserved in Bouin fixative. The fixed tissues were stained with Hematoxylin-Eosin, and then examined microscopically.
Postmortem examinations (offspring):
no data
Statistics:
The data were analyzed for homogeneity of variance using F test since the number of compared groups was two. If F test showed no statistical significance, the data were analyzed by Student's t test. If F test showed statistical significance, the data were subjected to Welch's t test. Date such as gross findings presented as frequencies were analyzed by square x-test followed by the Fisher's exact test to compare the vehicle a control group and treatment group where necessary. A difference was considered statistically significant at p<0.05 or p<0.01. Statistical analyses were performed by comparing the treatment groups with the vehicle control group using Path/Tox System and Statistical Analysis Systems (SAS/STAT User's Guide Version 8.2, NC, USA).
Reproductive indices:
Based on the results, following indices were calculated:
1) Mating index = (No. of animals with successful copulation / No. of mated animals) x 100
2) Fertility index = (No. of impregnation animals / No. of animals with successful copulation) x 100
3) Pregnancy index = (No. of pregnant animals / No. of animals with successful copulation) x 100
4) Delivery index: No. of dams with live newborns / No. of pregnant dams
5) Pre-implantation loss = ((No. of corpora lutea - No. of implantation) / No. of corpora lutea) x 100
6) Post-implantation loss = ((No. of implantation - litter size) / No. of implantation) x 100
7) Death rate of neonates to implantations = (No. of dead pups at birth / No. of implantation sites) x 100
Offspring viability indices:
- Viability index = (No. of live pups on Day 4 of lactation / No. of neonates at birth) x 100
- Sex ratio = No. of male newborns / No. of female newborns

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
During the premating period, bite wound was observed in 1 male of the vehicle control. During the mating period, loss of fur and bite wound were observed in 2 and 1 males in the vehicle control group, and scratched wound was observed in 1 male of the 1,000 mg/kg group. In females, no treatment-related clinical sign was observed in any groups.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no statistically significant changes in body weights of the treatment group.
No change in food consumption was observed in the treatment group.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No statistically significant change in weights of testis and epididymis was observed in the treatment group.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- time course of mating: there were no statistically significant differences in the mean time taken to mate in the treatment group.
- fertility and mating data: no treatment-related changes were observed in the copulation, fertility and pregnancy indices.
- no treatment-related effect was seen in all treatment groups in the following parameters examined: gestation length, the number of corpora lutea, implantation sites, delivery index, and pre- and post-implantation losses.

GROSS PATHOLOGY (PARENTAL ANIMALS)
At the necropsy, no abnormal finding was observed in any groups.

HISTOPATHOLOGY (PARENTAL ANIMALS)
During histopathological examination of males, tubular cell vacuolation of seminiferous tubule and inflammatory cell foci of prostate were observed in 2 males each and 4 males each in the 0 and 1,000 mg/kg group, respectively. Inflammatory cell foci of epididymis was observed in 3 males of the vehicle control group, and tubular atrophy of seminiferous tubule, olgospermia and cellular debris of epididymis was observed in 1 male of the 1,000 mg/kg group. In females, there was no abnormal finding in any groups.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not specified

Details on results (F1)

VIABILITY (OFFSPRING)
There were no treatment-related changes in the neonatal death or viability index.

BODY WEIGHT (OFFSPRING)
No treatment-related effect was seen in any of the treatment groups in body weights of pups in both sexes on Day 0 and 4 of lactation.

GROSS PATHOLOGY (OFFSPRING)
During the observation of live pups at birth, there were no externally malformed pups in any groups. At necropsy of pups, no gross finding was observed in any groups.

OTHER FINDINGS (OFFSPRING)
There were no treatment-related changes in the litter size at birth, or sex ratio of neonates.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
This study found no indication of any reproductive toxicity in parent animals at the maximum tested dose of 1,000 mg/kg bw/day. Therefore, the NOAEL for reproductive toxicity was 1,000 mg/kg bw/day.
Executive summary:

The test item, titanium dioxide (CAS No. 13463-67-7), was given orally at dose levels of 0 and 1,000 mg/kg bw/day (limit test) to Sprague-Dawley male rats from 2 weeks before mating to the end of the mating period, and to females from 2 weeks before mating to day 3 of lactation including the gestation period. Ten males and 10 females were used in each group. Effects of the test item in general findings and reproductive development were examined. The results obtained in this study were summarized as follow:

1) Effects on general findings: No treatment-related changes were observed in clinical signs, body weight, food consumption, gross finding and histopathological finding of parental rats.

2) Effects on reproductive performance: There were no treatment-related changes in pre-coital time, copulation index, fertility index and pregnant index in any of treatment groups. Furthermore, there were no treatment-related changes in the number of corpora lutea and implantation, pre-implantation loss, and post-implantation loss.

3) Effects on F1 offspring: There were no treatment-related changes in the litter size at birth, neonatal death, sex ratio of neonates, viability index, body weights of neonates on Day 0 and 4 of lactation or gross finding at any of the doses tested.

Based on the results, it was concluded that the oral administration of titanium dioxide to parent animals resulted in no all parameters examined at 1,000 mg/kg bw/day. Therefore, no observed adverse effect levels (NOAEL) of the test item are considered to be above 1,000 mg/kg bw/day in both sexes for general toxicity, reproductive capability and F1 neonates.