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Developmental toxicity / teratogenicity

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Endpoint:
developmental toxicity
Remarks:
90-day repeated dose oral toxicity study with information about developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
The reference exhibits major reporting and experimental deficiencies, which do not allow an independent review about the exposure-effects correlation: - purity of the test item was missing - animals were too old at the start of dosing. The guideline foresees animals that males are not older than about five to nine weeks of age. In this study, the animals were about 14 weeks old at the start of dosing. - number of pregnant females too low. The guideline foresees that about about 20 pregnant females are contained in each dose/control group. In this study a total of nine females were pregnant and it was not clear to which dose groups the females belonged. - the guideline foresees that at least three dose levels will be used. In this study two dose levels were only investigated, which precludes the possibility to determine a No-Observed-Adverse Effect level (NOAEL). - dosing on a 5-day basis only (normally 7-day basis). According to test guideline, the test substance should be administered daily from implantation to the day prior sacrifice to observe potential developmental effects. - body weight of male animals at the start of the study is questionable. According to the author, male and female animals weighed about 200 g at the start of the study. A figur showing the body weight development of the animals, starting on treatment day 8, suggested that the males gained about 100 g body weight in eight days (body weight of males about 300 g on treatment day 8), which seems unlikely. - mating procedure not described - details on environmental conditions for animals are not reported - acclimation period not stated - justification for choice of administration route not available - animals were not weighed as described in die guideline. The guideline foresees that females should be weighed at least every 3 days druing the dosing period. - food consumption were not recorded at three-day intervals. - females were not killed one day prior delivery for caesarean section and examination of all unborn pups. All females gave regular birth on gestation day 23. - uterine contents were not axamined. No information available on gravid uteri weight, number of corpora lutea, uterine contents, number of implantation sites and resorptions. - no data available on sex and litter/body weight of foetus - no data on external alterations and skeletal and soft tisse alterations. - information on observations of clinical signs and mortality is incomplete - historical control data and individual data are missing. Without the inclusion of historical control data it is impossible to determine, if the findings were test item-related effects or if the findings were still in the normal range for the species/strain used.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Several methodological deficiencies from the OECD 408 (1981) guideline were noted: purity of the test item missing; the OECD 408 (1981) guideline foresees that at least 20 animals (10 females /10 males) will be used at each dose level during a subchronic study. In this study, one dose group included 14 males / 5 females and another dose group included 5 males / 10 females, while the control group contained only 6 animals per sex; animals were too old at the start of dosing. The guideline foresees animals that are not older than 8 weeks of age. In this study, the animals were about 14 weeks old at the start of dosing; the guideline foresees that at least three dose levels will be used. In this study only two dose levels were investigated; body weight of male animals at the start of the study is questionable; information on observations of clinical signs and mortality is incomplete. It is uncertain how often the animals were observed; the guideline foresees the following observations: opthalmological examinations, haematology, clinical chemistry, organ weights, gross pathology and histopathology. The opthalmological examinations are completely missing and haematology (haematocrit and measurement of clotting potential missing), clinical chemistry, organ weights (adrenals and testes missing) and histopathology are incomplete; animals were not immediately sacrificed at the end of the study, but within a week after test item treatment ceased; gross pathology is not clearly described in the reference; Historical control data and individual data are missing; housing conditions are not fully described.
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Chromium (III) oxide was administered to groups of male and female BD rats via feed at concentrations of 2 % (14 males / 5 females) and 5 % (5 males / 10 females)(equivalent to 1000 and 2500 mg/kg bw; nominal concentrations). The test item was integrated in bread that was fed to the rats on 5 days/week during a study duration of 90 days. An untreated control group (6 males / 6 females) was run concurrently. The following parameters were investigated/recorded: clinical signs, mortality, body weights, food consumption, compound intake, haematology, clinical chemistry, urinalysis, gross pathology and histopathology.
GLP compliance:
not specified
Remarks:
not specified in this publication
Limit test:
no
Specific details on test material used for the study:
not specified
Species:
rat
Strain:
other: inbred BD
Details on species / strain selection:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: about 100 days old
- Weight at study initiation (average): about 200 g
- Housing: housed in groups of four in Makrolon cages
- Diet (control group): Altromin®
- Water (ad libitum): tap water
Route of administration:
oral: feed
Details on route of administration:
not specified
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
- DIET PREPARATION
The test item was administered in bread, which was made twice weekly by incorporating 2 or 5 % Cr2O3 into a dough made from 2835 g flour, 150 g milk powder, 150 g mixed salts (NaCl, CaHPO4 and Cu, Zn. Co, Mn and K), 150 g cooking oil, 150 g cod-liver oil, 300 g malt extract, 600 g sugar, 80 g yeast and 900 mL water and subsequently baked. Uneaten bread was weighed in order to determine the amount consumed. There were only small losses from crumbs.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
5 days/week
Dose / conc.:
2 other: % (nominal in diet)
Remarks:
equivalent to 1000 mg/kg bw (nominal in diet)
Dose / conc.:
5 other: % (nominal in diet)
Remarks:
equivalent to 2500 mg/kg bw (nominal in diet)
No. of animals per sex per dose:
2 % dose level: 14 males / 5 females
5 % dose level: 5 males / 10 females
Control animals:
yes, concurrent no treatment
Details on study design:
The animals of the treatment group received the control diet with a vegetable supplement at the weekends.
Positive control:
not specified
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked: clinical signs and mortality

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: every eighth day

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was determined: Yes , weekly
- Compound intake determined: Yes, uneaten bread was weighed in order to determine the amount consumed. There were only small losses from crumbs.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE: Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before the beginning of the experiment, monthly and at the end of the feeding period
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, 24 hours (measurement at the end of the feeding period only)
- How many animals: all animals
- Parameters examined: haemaglobin, erythrocyte count as well as, total and differential 8neutrophils, lymphocytes, monocytes and eosiniphils) leucocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before the beginning of the experiment, monthly and at the end of the feeding period
- Animals fasted: Yes, 24 hours (measurement at the end of the feeding period only)
- How many animals: all animals
- Parameters examined: blood sugar, serum protein and serum bilirubin

URINALYSIS: Yes
- Time schedule for collection of urine: random samples were taken during the course of the study and from all animals in the last week of the experiment
- Metabolism cages used for collection of urine: Not specified
- Parameters examined: protein, sugar, bilirubin, blood and sediment

NEUROBEHAVIOURAL EXAMINATION: Not specified
IMMUNOLOGY: Not specified
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
All the experimental animals except those from the fertility study, which were still suckling, were killed within 1 week of the cessation of Cr2O3 feeding.
An autopsy on all major organs was performed. The liver, spleen and kidney were weighed for all animals and the brain and ovaries were weighed in randomly selected animals. Samples of these organs and of lung, heart, pancreas, stomach, small intestine and urinary bladder were fixed in 4 % buffered formalin and sections were embedded in paraffin wax and stained with haematoxylin and eosin.
Other examinations:
During the last 30 days of treatment, males and females from the same group were paried to test fertility, and the number of young in each liiter was recorded.
Statistics:
mean ± SEM
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
CLINICAL SIGNS:
- 2 and 5 % dose levels: treatment was well tolerated at both dose levels.
The faeces of the treated animals showed an intense green colouration throughout the study.

MORTALITY:
- 0 and 2 % dose levels: no mortality was observed.
- 5 % dose level: 1/5 male died 70 days after the start of the experiment from acute pneumonia.

BODY WEIGHTS:
0, 2 and 5 % dose levels: no significant differences in body weight were evident between the two experimental groups and the controls.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- food intake was normal throughout the study, averaging 20 g/day for males and 15 g/day for females.
- 2 % dose level: total amount of Cr2O3 consumed during the whole study was 75 g/kg bw (25 g/animal) for males and 72 g/kg bw (18 g/animal) for females
- 5 % dose level: total amount of Cr2O3 consumed during the whole study was 180 g/kg bw for males and 160 g/kg bw for females

HAEMATOLOGICAL FINDINGS (findings of the determination conducted at the end of the feeding period were only presented):
- 2 and 5 % dose levels: haemaglobin determinations and counts of erythrocytes and total and differential leucocytes in blood samples of the treated animals showed no statistically siginficant deviations from the corresponding control values.

CLINICAL BIOCHEMISTRY FINDINGS (findings of the determination conducted at the end of the feeding period were only presented):
- 2 and 5 % dose levels: there were no adverse findings in determinations of serum protein, serum bilirubin and blood sugar (no statstically significant differences from control was found). The slightly reduced total bilirubin content in serum was within the noraml limits for the strain of rat used.

URINALYSIS FINDINGS:
- 2 and 5 % dose levels: analysis cariied out during the eyperiment and at termination showed no statsitcally significant differences between the treated and control animals.

ORGAN WEIGHT FINDINGS:
- 2 and 5 % dose levels: the weights of the spleen and liver showed some reduction in the treated animals as follows (dose-sependent):
Spleen (males):
control group: 0.82 ± 0.08 g
2 % dose level: 0.61 ± 0.15 g
5 % dose level: 0.52 ± 0.12 g

Liver (males):
control group: 8.9 ± 0.7 g
2 % dose level: 8.8 ± 0.6 g
5 % dose level: 7.7 ± 0.5 g

Spleen (females):
control group: 0.55 ± 0.10 g
2 % dose level: 0.48 ± 0.16 g
5 % dose level: 0.43 ± 0.15 g

Liver (females):
control group: 7.3 ± 0.5 g
2 % dose level: 6.7 ± 0.7 g
5 % dose level: 5.1 ± 0.6 g

No differences were noticed in the remaining organs.

GROSS PATHOLOGICAL FINDINGS:
- 2 and 5 % dose levels: there were no macroscopic changes.

HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
- 2 and 5 % dose levels: there were no histological changes. Compared with sections of organs from control animals, no changes could be detected that could be attributed to the Cr2O3 treatment.
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
not specified
Conclusions:
Ivankovic & Preussmann (1975) investigated the repeated dose oral toxicitiy of chromium (III) oxide in male and female BD rats, which received the substance via feed at concentrations of 2 % (14 males / 5 females) and 5 % (5 males / 10 females)(equivalent to 1000 and 2500 mg/kg bw).The animals received the substance 5 days/week for a study duration of 90 days. An untreated control group (6 males / 6 females) was run concurrently. No test item related effects were observed for clinical signs, mortality, body weights, food consumption, haematological findings, clinical chemistry findings, urinalysis, macroscopical examinations and histopathological findings. During organ weigth examinations the absolute weight of the spleen and the liver showed some reduction in the treated male and female animals. The organ weight findings were not considered to be test item-related, since no findings for these organs were made during gross pathologiy or histopathology.

The reference exhibits major reporting and experimental deficiencies, which do not allow an independent review about the exposure-effects correlation:

- the OECD guideline 408 (1981) foresees that at least 20 animals (10 females /10 males) will be used at each dose level during a subchronic study. In this study, one dose group included 14 males / 5 females and another dose group included 5 males / 10 females, while the control group contained only 6 animals per sex.
- animals were too old at the start of dosing. The guideline foresees animals that are not older than 8 weeks of age. In this study, the animals were about 14 weeks old at the start of dosing.
- the guideline foresees that at least three dose levels will be used. In this study two dose levels were only investigated, which precludes the possibility to determine a No-Observed-Adverse Effect level (NOAEL).
- body weight of male animals at the start of the study is questionable. According to the author, male and female animals weighed about 200 g at the start of the study. A figur showing the body weight development of the animals, starting on treatment day 8, suggested that the males gained about 100 g body weight in eight days (body weight of males about 300 g on treatment day 8), which seems unlikely.
- information on observations of clinical signs and mortality is incomplete. It is uncertain how often the animals were observed. A daily observations of the clinical signs is important in order not to miss any toxicological effects caused by the test substance.
- the guideline foresees the following observations: opthalmological examinations, haematology, clinical chemistry, organ weights, gross pathology and histopathology. The opthalmological examinations are completely missing and haematology (haematocrit and measurement of clotting potential missing), clinical chemistry, organ weights (adrenals and testes missing) and histopathology are incomplete.
- animals were not immediately sacrificed at the end of the study, but within a week after test item treatment ceased.
- gross pathology is not clearly described in the reference.
- historical control data and individual data are missing. Without the inclusion of historical control data it is impossible to determine, if the findings were test item-related effects or if the findings were still in the normal range for the species/strain used.
- no analytical verification of the dose levels in feed was conducted. In addition the homogeneity and stability in feed was not determined.
- housing conditions are not fully described.
Reason / purpose:
reference to same study
Reference
Endpoint:
fertility, other
Remarks:
90-day repeated dose oral toxicity study with information about fertility and offspring
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Several methodological deficiencies from the OECD 415 (1983) guideline were noted: purity of the test item missing; animals were too old at the start of dosing; number of pregnant females too low; two dose levels tested only; dosing on a 5-day basis only (normally 7-day basis); dosing schedule not according to guideline; mating procedure not described; incomplete litter observations (sex, number of stillbirths, number of live births, number of dead pups, litter/pup weight missing); unclear if macroscopic/microscopic examinations of reproductive organs were conducted; body weight of male animals at the start of the study is questionable; information on observations of clinical signs and mortality is incomplete; fertility, gestation and viability indices missing; individual data missing; historical control data missing.
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Chromium (III) oxide was administered to groups of male and female BD rats via feed at concentrations of 2 % (14 males / 5 females) and 5 % (5 males / 10 females)(equivalent to 1000 and 2500 mg/kg bw; nominal concentrations). The test item was integrated in bread that was fed to the rats on 5 days/week during a study duration of 90 days. An untreated control group (6 males / 6 females) was run concurrently. The following parameters were investigated/recorded: clinical signs, mortality, body weights, food consumption, compound intake, haematology, clinical chemistry, urinalysis, gross pathology and histopathology. In addition, during the last 30 days of treatment, males and females were paired to test fertility, and the number of young in each litter was recorded.
GLP compliance:
not specified
Remarks:
not specified in this publication
Limit test:
no
Specific details on test material used for the study:
not specified
Species:
rat
Strain:
other: inbred BD
Details on species / strain selection:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: about 100 days old
- Weight at study initiation (average): about 200 g
- Housing: housed in groups of four in Makrolon cages
- Diet (control group): Altromin®
- Water (ad libitum): tap water
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
- DIET PREPARATION
The test item was administered in bread, which was made twice weekly by incorporating 2 or 5 % Cr2O3 into a dough made from 2835 g flour, 150 g milk powder, 150 g mixed salts (NaCl, CaHPO4 and Cu, Zn. Co, Mn and K), 150 g cooking oil, 150 g cod-liver oil, 300 g malt extract, 600 g sugar, 80 g yeast and 900 mL water and subsequently baked. Uneaten bread was weighed in order to determine the amount consumed. There were only small losses from crumbs.
Details on mating procedure:
Altogether nine females were paired with males from the same dose group 60 days after the start of feeding.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
5 days/week
Dose / conc.:
2 other: % (nominal in diet)
Remarks:
equivalent to 1000 mg/kg bw (nominal in diet)
Dose / conc.:
5 other: % (nominal in diet)
Remarks:
equivalent to 2500 mg/kg bw (nominal in diet)
No. of animals per sex per dose:
2 % dose level: 14 males / 5 females
5 % dose level: 5 males / 10 females
Control animals:
yes, concurrent no treatment
Details on study design:
The animals of the treatment group received the control diet with a vegetable supplement at the weekends.
Positive control:
not specified
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked: clinical signs and mortality

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: every eighth day

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was determined: Yes, weekly
- Compound intake determined: Yes, uneaten bread was weighed in order to determine the amount consumed. There were only small losses from crumbs.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE: Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before the beginning of the experiment, monthly and at the end of the feeding period
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, 24 hours (measurement at the end of the feeding period only)
- How many animals: all animals
- Parameters examined: haemaglobin, erythrocyte count as well as, total and differential 8neutrophils, lymphocytes, monocytes and eosiniphils) leucocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before the beginning of the experiment, monthly and at the end of the feeding period
- Animals fasted: Yes, 24 hours (measurement at the end of the feeding period only)
- How many animals: all animals
- Parameters examined: blood sugar, serum protein and serum bilirubin

URINALYSIS: Yes
- Time schedule for collection of urine: random samples were taken during the course of the study and from all animals in the last week of the experiment
- Metabolism cages used for collection of urine: Not specified
- Parameters examined: protein, sugar, bilirubin, blood and sediment

NEUROBEHAVIOURAL EXAMINATION: Not specified
IMMUNOLOGY: Not specified

OTHER OBSERVATIONS:
- duration of gestation
Oestrous cyclicity (parental animals):
not specified
Sperm parameters (parental animals):
not specified
Litter observations:
The number of young was recorded and examined for adverse effects following exposure of parental animals
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
All the experimental animals except those from the fertility study, which were still suckling, were killed within 1 week of the cessation of Cr2O3 feeding.
An autopsy on all major organs was performed. The liver, spleen and kidney were weighed for all animals and the brain and ovaries were weighed in randomly selected animals. Samples of these organs and of lung, heart, pancreas, stomach, small intestine and urinary bladder were fixed in 4 % buffered formalin and sections were embedded in paraffin wax and stained with haematoxylin and eosin.
Postmortem examinations (offspring):
Clinical signs were recorded for the offspring.
Offspring were macroscopically investigated.
Some of the progeny were retained for lifetime observations (600 days) in order to investigate the tumour formation.
Statistics:
mean ± SEM
Reproductive indices:
not specified
Offspring viability indices:
not specified
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
CLINICAL SIGNS:
- 2 and 5 % dose levels: treatment was well tolerated at both dose levels.
The faeces of the treated animals showed an intense green colouration throughout the study.

MORTALITY:
- 0 and 2 % dose levels: no mortality was observed.
- 5 % dose level: 1/5 male died 70 days after the start of the experiment from acute pneumonia.

BODY WEIGHTS:
0, 2 and 5 % dose levels: no significant differences in body weight were evident between the two experimental groups and the controls.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- food intake was normal throughout the study, averaging 20 g/day for males and 15 g/day for females.
- 2 % dose level: total amount of Cr2O3 consumed during the whole study was 75 g/kg bw (25 g/animal) for males and 72 g/kg bw (18 g/animal) for females
- 5 % dose level: total amount of Cr2O3 consumed during the whole study was 180 g/kg bw for males and 160 g/kg bw for females

HAEMATOLOGICAL FINDINGS (findings of the determination conducted at the end of the feeding period were only presented):
- 2 and 5 % dose levels: haemaglobin determinations and counts of erythrocytes and total and differential leucocytes in blood samples of the treated animals showed no statistically siginficant deviations from the corresponding control values.

CLINICAL BIOCHEMISTRY FINDINGS (findings of the determination conducted at the end of the feeding period were only presented):
- 2 and 5 % dose levels: there were no adverse findings in determinations of serum protein, serum bilirubin and blood sugar (no statstically significant differences from control was found). The slightly reduced total bilirubin content in serum was within the noraml limits for the strain of rat used.

URINALYSIS FINDINGS:
- 2 and 5 % dose levels: analysis cariied out during the eyperiment and at termination showed no statsitcally significant differences between the treated and control animals.

ORGAN WEIGHT FINDINGS:
- 2 and 5 % dose levels: the weights of the spleen and liver showed some reduction in the treated animals as follows (dose-sependent):
Spleen (males):
control group: 0.82 ± 0.08 g
2 % dose level: 0.61 ± 0.15 g
5 % dose level: 0.52 ± 0.12 g

Liver (males):
control group: 8.9 ± 0.7 g
2 % dose level: 8.8 ± 0.6 g
5 % dose level: 7.7 ± 0.5 g

Spleen (females):
control group: 0.55 ± 0.10 g
2 % dose level: 0.48 ± 0.16 g
5 % dose level: 0.43 ± 0.15 g

Liver (females):
control group: 7.3 ± 0.5 g
2 % dose level: 6.7 ± 0.7 g
5 % dose level: 5.1 ± 0.6 g

No differences were noticed in the remaining organs.

GROSS PATHOLOGICAL FINDINGS:
- 2 and 5 % dose levels: there were no macroscopic changes.

HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
- 2 and 5 % dose levels: there were no histological changes. Compared with sections of organs from control animals, no changes could be detected that could be attributed to the Cr2O3 treatment.

REPRODUCTIVE PERFORMANCE:
- 2 % and 5 % dose levels: all the females became pregnant in due course, the gestation period was normal (23 days) Litter sizes were in the normal range, average eight pups.Fertility was not adversely effected.
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
not specified
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
CLINICAL SIGNS:
- 2 % and 5 %: the young showed no adverse effects.

GROSS PATHOLOGY:
- 2 % and 5 %: the young showed no malformation.

HISTOPATHOLOGY
- 2 % and 5 %: no tumours have been detected.
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
not specified
Reproductive effects observed:
not specified
Conclusions:
Rats exposed to high doses of chromic oxide (2% and 5% dietary level) in an early subchronic feeding study were paired to evaluate adverse effects on reproduction performance and offspring. Nine females were paired with males from the same dosage group 60 days after the start of feeding. All females became pregnant, the gestation period was normal (23 days) and the offspring showed no malformation or other adverse effects. Litter sizes were in the normal range (average eight pups). Some of the progeny were retained for lifetime observation and no tumours were detected.

The reference exhibits major reporting and experimental deficiencies, which do not allow an independent review about the exposure-effects correlation:

- purity of the test item was missing
- animals were too old at the start of dosing. The guideline foresees animals that males are not older than about five to nine weeks of age. In this study, the animals were about 14 weeks old at the start of dosing.
- number of pregnant females too low. The guideline foresees that about about 20 pregnant females are contained in each dose/control group. In this study a total of nine females were pregnant and it was not clear to which dose groups the females belonged.
- the guideline foresees that at least three dose levels will be used. In this study two dose levels were only investigated, which precludes the possibility to determine a No-Observed-Adverse Effect level (NOAEL).
- dosing on a 5-day basis only (normally 7-day basis), which could enable a recovery from the test item.
- dosing schedule not according to guideline. As described in the reference the animals were dosed 60 days before mating occurred. According to the guideline males should be dosed during growth and for at least one complete spermatogenic cycle (70 days in rat) in order to elicit any adverse effects on spermegonesis by the test item. Since the animals were only exposed to the test item for a duration of 60 days the complete spermatogenic cycle was not covered by test substance administration. Therefore, it can not be assumed that the effect on male fertility is complete investigated.
- mating procedure not described
- incomplete litter observations (sex, number of stillbirths, number of live births, number of dead pups, litter/pup weight missing)
- unclear if macroscopic/microscopic examinations of reproductive organs were conducted, which is important to investigate the endpoint fertility
- body weight of male animals at the start of the study is questionable. According to the author, male and female animals weighed about 200 g at the start of the study. A figur showing the body weight development of the animals, starting on treatment day 8, suggested that the males gained about 100 g body weight in eight days (body weight of males about 300 g on treatment day 8), which seems unlikely.
- information on observations of clinical signs and mortality is incomplete and fertility, gestation and viability indices are not determined
- historical control data and individual data are missing. Without the inclusion of historical control data it is impossible to determine, if the findings were test item-related effects or if the findings were still in the normal range for the species/strain used.
Reason / purpose:
reference to same study
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Several methodological deficiencies from the OECD 451 (1981) guideline were noted: the OECD guideline 451(1981) prefers that dosing of rodents beginn as soon as possible after weaning and acclimatisation, and preferable before the animals are 6 weeks old. The animals used in this study were about 14 weeks of age at start of the study; no analytical verification of the dose levels in feed was conducted. In addition the homogeneity and stability in feed was not determined; the guideline foresees that clinical signs are recorde at least once each day. The authors did not indicated how often the clinical signs were recorded as well as they did not stated how often mortality was reocrded; the guideline foresees that the body weight will be recorded once a week during the first 13 weeks of the test period and at least once every 4 weeks thereafter. In this study, the body weight of the animals were not recorded weekly during the first 13 weeks, but the body weight was recorded monthly through out the study; the guideline foresees that the food consumption will be determined, but in this study the food consumption determination was not described. It is assumed that it was recorded, since compound intake was determined; the guideline foresees that all organs and tissues of all animals should be preserved for microscopic evaluation. In this study the authors only indicates that all important organs incl. brain and nervous system were studied histologically. It is not quite clear which organs were part of the histological examination. In addition, macroscopic evaluation is apparently carried out, but the scope of this evaluation was not fully described.; the guideline foresees a differential blood count of the highest dose group. This was not done in the current study; historical control data and individual data are missing.
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Chromium (III) oxide was administered to groups of 60 male and 60 female BD rats via feed at concentrations of 1%, 2 % and 5 % (equivalent to 500, 1000 and 2500 mg/kg bw; nominal concentrations). The test item was integrated in bread that was fed to the rats on 5 days/week during a study duration of 2 years (600 feeding days). An untreated control group was run concurrently. The following parameters were investigated/recorded: clinical signs, mortality, body weights, compound intake, gross pathology and histopathology.
GLP compliance:
not specified
Remarks:
not specified in this publication
Specific details on test material used for the study:
not specified
Species:
rat
Strain:
other: inbred BD
Details on species / strain selection:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: about 100 days old
- Weight at study initiation (average): about 200 g
- Housing: housed in groups of four in Makrolon cages
- Diet (control group): Altromin®
- Water (ad libitum): tap water
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
- DIET PREPARATION
The test item was administered in bread, which was made twice weekly by incorporating 2 or 5 % Cr2O3 into a dough made from 2835 g flour, 150 g milk powder, 150 g mixed salts (NaCl, CaHPO4 and Cu, Zn. Co, Mn and K), 150 g cooking oil, 150 g cod-liver oil, 300 g malt extract, 600 g sugar, 80 g yeast and 900 mL water and subsequently baked. Uneaten bread was weighed in order to determine the amount consumed. There were only small losses from crumbs.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
2 years
Frequency of treatment:
5 days/week (600 feeding days)
Post exposure period:
At the end of the feeding period, surviving animals were maintained on the control diet until they died or became moribund, death being induced with ehter in the latter case.
Dose / conc.:
1 other: % (nominal in diet)
Remarks:
equivalent to 500 mg/kg bw (nominal in diet)
Dose / conc.:
2 other: % (nominal in diet)
Remarks:
equivalent to 1000 mg/kg bw (nominal in diet)
Dose / conc.:
5 other: % (nominal in diet)
Remarks:
equivalent to 2500 mg/kg bw (nominal in diet)
No. of animals per sex per dose:
60 males / 60 females
Control animals:
yes, concurrent no treatment
Details on study design:
The animals of the treatment group received the control diet with a vegetable supplement at the weekends.
Positive control:
not specified
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked: clinical signs and mortality

DETAILED CLINICAL OBSERVATIONS: No data
DERMAL IRRITATION: No data

BODY WEIGHT: Yes
- Time schedule for examinations: monthly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake: Yes
Intake of the test item was calculated weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data
WATER CONSUMPTION AND COMPOUND INTAKE: No data
OPHTHALMOSCOPIC EXAMINATION: No data
HAEMATOLOGY: No data
CLINICAL CHEMISTRY: No data
URINALYSIS: No data
NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
At autopsy, all the important organs, including the brain and nervous system, were fixed in 4 % formalin solution and studied histologically.
Statistics:
not specified
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Details on results:
CLINICAL SIGNS:
- 1, 2 and 5 % dose levels: feeding of Cr2O3 at all three dose levels was well tolerated.

MORTALITY:
- control group: the control rats reached an average life expectancy of 890 days (range: 860 - 935 days).
- 1 % dose level: the first died from a lung infection after 130 days and the last 1315 days' from the start of the experiment The average life expectancy was 870day (range: 855 - 900 days).
- 2 % dose level: the first death occurred after 127 days and the last 1230 days from the start of feeding. The average survival period was 880 days (range: 860 - 910 days.
- 5 % dose level: the first rat died after 113 days and the last after 1295 days. The average survival was 860 days (range: 835 - 900 days).

Overall, the median survival times in all three dosage groups were comparable with that of the controls.

COMPOUND INTAKE:
- 1 % dose level: total dose of Cr2O3 consumed was about 120 g/rat, or about 360 g/kg bw in 600 days of feeding.
- 2 % dose level: total dose of Cr2O3 consumed in 600 days of feeding was 240 g/rat, or 720 g/kg bw.
- 5 % dose level: total dose of Cr2O3 consumed in 600 days or feeding was about 600 g/animal or 1800 g/kg bw.

BODY WEIGHTS:
- 1, 2 and 5 % dose levels: the body weight curves for the treated animals did not differ from those for the controls.

GROSS PATHOLOGICAL FINDINGS:
- 1, 2 and 5 % dose levels: no macroscopic post-mortem findings could be causally related to the Cr2O3 treatment.

HISTOPATHOLOGY- NON-NEOPLASTIC
- 1, 2 and 5 % dose levels: no histological post-mortem findings could be causally related to the Cr2O3 treatment.

HISTOPATHOLOGY - NEOPLASTIC
- control group: two mammary fibroadenomas, one mammary carcinoma and two hypophyseal adenomas were observed in this group.
- 1 % dose level: only three mammary fibroadenomas were detected in this group. The remaining animals showed no macroscopic or microscopic signs of benign or malignant growths.
- 2 % dose level: one mammary fibroadenoma was the only tumour observed.
- 5 % dose level: three mammary fibroadenomas and one hypophyseal adenoma were found, but no other benign or malignant tumours appeared.

The type and frequency of the tumours appearing in the experimental and control animals were comparable. Mammary fibroadenomas and the hypophyseal adenomas are "spontaneous" tumours characteristic of the strains of BD rats.
Relevance of carcinogenic effects / potential:
The type and frequency of the tumours appearing in the experimental and control animals were comparable. Mammary fibroadenomas and the hypophyseal adenomas are "spontaneous" tumours characteristic of the strains of BD rats.
Critical effects observed:
not specified
Conclusions:
Ivankovic & Preussmann (1975) investigated the repeated dose oral toxicitiy of chromium (III) oxide in groups of 60 male and 60 female BD rats, which received the substance via feed at concentrations of 1 %, 2 % and 5 % (equivalent to 500, 1000 and 2500 mg/kg bw; nominal concentrations). The animals received the substance 5 days/week for a study duration of 2 years. An untreated control group was run concurrently. No test item-related effects were observed for clinical signs, mortality, body weight, macroscopical findings and histopathologcal findings. In the control group and experimental groups mammary fibroadenomas and hypophyseal adenomas were found. These tumours are considered to be characteristic for the rat strain and therefore are not considered to be induced by the test item.

The reference exhibits major reporting and experimental deficiencies, which do not allow an independent review about the exposure-effects correlation:

- the OECD guideline 451 (1981) prefers that dosing of rodents beginn as soon as possible after weaning and acclimatisation, and preferable before the animals are 6 weeks old. The animals used in this study were about 14 weeks of age at start of the study.
- no analytical verification of the dose levels in feed was conducted. In addition the homogeneity and stability in feed was not determined.
- the guideline foresees that clinical signs are recorde at least once each day. The authors did not indicated how often the clinical signs were recorded as well as they did not stated how often mortality was recorded.
- the guideline foresees that the body weight will be recorded once a week during the first 13 weeks of the test period and at least once every 4 weeks thereafter. In this study, the body weight of the animals were not recorded weekly during the first 13 weeks, but the body weight was recorded monthly through out the study.
- the guideline foresees that the food consumption will be determined, but in this study the food consumption determination was not described. It is assumed that it was recorded, since compund intake was determined.
- the guideline foresees that all organs and tissues of all animals should be preserved for microscopic evaluation. In this study the authors only indicates that all important organs incl. brain and nervous system were studied histologically. It is not quite clear which organs were part of the histological examination. In addition, macroscopic evaluation is apparently carried out, but the scope of this evaluation was not fully described.
- the guideline foresees a differential blood count of the highest dose group. This was not done in the current study.
- historical control data and individual data are missing.

Based on the shortcomings given above, this publication was disregarded for the hazard and risk assessment.

Data source

Reference
Reference Type:
publication
Title:
Absence of toxic and carcinogenic effects after administration of high doses of chromic oxide pigment in subacute and long-term feeding experiments in rats
Author:
Ivankovic S, Preussmann R.
Year:
1975
Bibliographic source:
Fd Cosmet Toxicol 13: 347-351

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Chromium (III) oxide was administered to groups of male and female BD rats via feed at concentrations of 2 % (14 males / 5 females) and 5 % (5 males / 10 females)(equivalent to 1000 and 2500 mg/kg bw; nominal concentrations). The test item was integrated in bread that was fed to the rats on 5 days/week during a study duration of 90 days. An untreated control group (6 males / 6 females) was run concurrently. The following parameters were investigated/recorded: clinical signs, mortality, body weights, food consumption, compound intake, haematology, clinical chemistry, urinalysis, gross pathology and histopathology. In addition, during the last 30 days of treatment, males and females were paired to test fertility, and the number of young in each litter was recorded.
GLP compliance:
not specified
Remarks:
not specified in this publication
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Chromium oxide green (chromium (III) oxide; Schultz no. 1451; CI no. 77288; pigment green 17) was obtained by reduction of chromate at about 600"C. The product, which was washed until all soluble components, particularly chromate, had disappeared, was a fine green powder of pure Cr2O3, insoluble in water, alkali and mineral acids. The product was in a non-hydrated form. Precise analysis of the preparation used in the study gave a maximum content of 3 ppm Cr3+. The preparation was free from chromate (detection limits of the method of determination) 1 μg CrO4²-). With respect to heavy-metal impurities (arsenic. lead, mercury, antimony, soluble barium, copper and zinc), the product complied with the specifications of the Deutsche Forschungsgemeinschaft, Farbstoff-Kommission (1959)*.

*Reference:
- Deutsche Forschungsgemeinschaft. Farbstoff-Kommission (1959). Mitteilung 3: Vorläufige Liste von Färbemitteln fur Kosmetika. 2nd Ed. (and new introductory text. 1962). Franz Steiner Verlag GmbH. Wiesbaden.
Specific details on test material used for the study:
not specified

Test animals

Species:
rat
Strain:
other: inbred BD
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: about 100 days old
- Weight at study initiation (average): about 200 g
- Housing: housed in groups of four in Makrolon cages
- Diet (control group): Altromin®
- Water (ad libitum): tap water

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
- DIET PREPARATION
The test item was administered in bread, which was made twice weekly by incorporating 2 or 5 % Cr2O3 into a dough made from 2835 g flour, 150 g milk powder, 150 g mixed salts (NaCl, CaHPO4 and Cu, Zn. Co, Mn and K), 150 g cooking oil, 150 g cod-liver oil, 300 g malt extract, 600 g sugar, 80 g yeast and 900 mL water and subsequently baked. Uneaten bread was weighed in order to determine the amount consumed. There were only small losses from crumbs.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Altogether nine females were paired with males from the same dose group 60 days after the start of feeding.
Duration of treatment / exposure:
90 days
Frequency of treatment:
5 days/week
Duration of test:
Nine females were paired with males from the same dose group following treatment for 60 days (no additional data given)
Doses / concentrationsopen allclose all
Dose / conc.:
2 other: % (nominal in diet)
Remarks:
equivalent to 1000 mg/kg bw (nominal in diet)
Dose / conc.:
5 other: % (nominal in diet)
Remarks:
equivalent to 2500 mg/kg bw (nominal in diet)
No. of animals per sex per dose:
2 % dose level: 14 males / 5 females
5 % dose level: 5 males / 10 females
Control animals:
yes, concurrent no treatment
Details on study design:
The animals of the treatment group received the control diet with a vegetable supplement at the weekends.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked: clinical signs and mortality

DETAILED CLINICAL OBSERVATIONS: Not specified
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: every eighth day

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was determined: Yes, weekly
- Compound intake determined: Yes, uneaten bread was weighed in order to determine the amount consumed. There were only small losses from crumbs.

WATER CONSUMPTION AND COMPOUND INTAKE: Not specified

POST-MORTEM EXAMINATIONS

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before the beginning of the experiment, monthly and at the end of the feeding period
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, 24 hours (measurement at the end of the feeding period only)
- How many animals: all animals
- Parameters examined: haemaglobin, erythrocyte count as well as, total and differential 8neutrophils, lymphocytes, monocytes and eosiniphils) leucocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before the beginning of the experiment, monthly and at the end of the feeding period
- Animals fasted: Yes, 24 hours (measurement at the end of the feeding period only)
- How many animals: all animals
- Parameters examined: blood sugar, serum protein and serum bilirubin

URINALYSIS: Yes
- Time schedule for collection of urine: random samples were taken during the course of the study and from all animals in the last week of the experiment
- Metabolism cages used for collection of urine: Not specified
- Parameters examined: protein, sugar, bilirubin, blood and sediment

NEUROBEHAVIOURAL EXAMINATION: Not specified
IMMUNOLOGY: Not specified

OTHER OBSERVATIONS:
- duration of gestation
Ovaries and uterine content:
not specified
Fetal examinations:
The number of young was recorded and examined for adverse effects following exposure of parental animals
Statistics:
mean ± SEM
Indices:
not specified
Historical control data:
not specified

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
not specified
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
not specified
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Details on maternal toxic effects:
CLINICAL SIGNS:
- 2 and 5 % dose levels: treatment was well tolerated at both dose levels.
The faeces of the treated animals showed an intense green colouration throughout the study.

MORTALITY:
- 0 and 2 % dose levels: no mortality was observed.
- 5 % dose level: 1/5 male died 70 days after the start of the experiment from acute pneumonia.

BODY WEIGHTS:
0, 2 and 5 % dose levels: no significant differences in body weight were evident between the two experimental groups and the controls.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- food intake was normal throughout the study, averaging 20 g/day for males and 15 g/day for females.
- 2 % dose level: total amount of Cr2O3 consumed during the whole study was 75 g/kg bw (25 g/animal) for males and 72 g/kg bw (18 g/animal) for females
- 5 % dose level: total amount of Cr2O3 consumed during the whole study was 180 g/kg bw for males and 160 g/kg bw for females

HAEMATOLOGICAL FINDINGS (findings of the determination conducted at the end of the feeding period were only presented):
- 2 and 5 % dose levels: haemaglobin determinations and counts of erythrocytes and total and differential leucocytes in blood samples of the treated animals showed no statistically siginficant deviations from the corresponding control values.

CLINICAL BIOCHEMISTRY FINDINGS (findings of the determination conducted at the end of the feeding period were only presented):
- 2 and 5 % dose levels: there were no adverse findings in determinations of serum protein, serum bilirubin and blood sugar (no statstically significant differences from control was found). The slightly reduced total bilirubin content in serum was within the noraml limits for the strain of rat used.

URINALYSIS FINDINGS:
- 2 and 5 % dose levels: analysis cariied out during the eyperiment and at termination showed no statsitcally significant differences between the treated and control animals.

ORGAN WEIGHT FINDINGS:
- 2 and 5 % dose levels: the weights of the spleen and liver showed some reduction in the treated animals as follows (dose-sependent):
Spleen (males):
control group: 0.82 ± 0.08 g
2 % dose level: 0.61 ± 0.15 g
5 % dose level: 0.52 ± 0.12 g

Liver (males):
control group: 8.9 ± 0.7 g
2 % dose level: 8.8 ± 0.6 g
5 % dose level: 7.7 ± 0.5 g

Spleen (females):
control group: 0.55 ± 0.10 g
2 % dose level: 0.48 ± 0.16 g
5 % dose level: 0.43 ± 0.15 g

Liver (females):
control group: 7.3 ± 0.5 g
2 % dose level: 6.7 ± 0.7 g
5 % dose level: 5.1 ± 0.6 g

No differences were noticed in the remaining organs.

GROSS PATHOLOGICAL FINDINGS:
- 2 and 5 % dose levels: there were no macroscopic changes.

HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
- 2 and 5 % dose levels: there were no histological changes. Compared with sections of organs from control animals, no changes could be detected that could be attributed to the Cr2O3 treatment.

REPRODUCTIVE PERFORMANCE:
- 2 % and 5 % dose levels: all the females became pregnant in due course, the gestation period was normal (23 days) Litter sizes were in the normal range, average eight pups.Fertility was not adversely effected.

Effect levels (maternal animals)

Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Abnormalities:
not specified

Results (fetuses)

Fetal body weight changes:
not specified
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not specified
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
EXTERNAL MALFORMATIONS:
The young showed no malformations or other adverse effects.

CHANCES IN LITTER SIZE AND WEIGHTS:
Litter sizes were in the normal range, average eight pups.

HISTOPATHOLOGY:
No tumors have been detected.

Effect levels (fetuses)

Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Rats exposed to high doses of chromic oxide (2% and 5% dietary level) in an early subchronic feeding study were paired to evaluate adverse effects on reproduction performance and offspring. Nine females were paired with males from the same dosage group 60 days after the start of feeding. All females became pregnant, the gestation period was normal (23 days) and the offspring showed no malformation or other adverse effects. Litter sizes were in the normal range (average eight pups). Some of the progeny were retained for lifetime observation and no tumours were detected.


The reference exhibits major reporting and experimental deficiencies, which do not allow an independent review about the exposure-effects correlation:

- purity of the test item was missing
- animals were too old at the start of dosing. The guideline foresees animals that males are not older than about five to nine weeks of age. In this study, the animals were about 14 weeks old at the start of dosing.
- number of pregnant females too low. The guideline foresees that about about 20 pregnant females are contained in each dose/control group. In this study a total of nine females were pregnant and it was not clear to which dose groups the females belonged.
- the guideline foresees that at least three dose levels will be used. In this study two dose levels were only investigated, which precludes the possibility to determine a No-Observed-Adverse Effect level (NOAEL).
- dosing on a 5-day basis only (normally 7-day basis). According to test guideline, the test substance should be administered daily from implantation to the day prior sacrifice to observe potential developmental effects.
- body weight of male animals at the start of the study is questionable. According to the author, male and female animals weighed about 200 g at the start of the study. A figur showing the body weight development of the animals, starting on treatment day 8, suggested that the males gained about 100 g body weight in eight days (body weight of males about 300 g on treatment day 8), which seems unlikely.
- mating procedure not described
- details on environmental conditions for animals are not reported
- acclimation period not stated
- justification for choice of administration route not available
- animals were not weighed as described in die guideline. The guideline foresees that females should be weighed at least every 3 days druing the dosing period.
- food consumption were not recorded at three-day intervals.
- females were not killed one day prior delivery for caesarean section and examination of all unborn pups. All females gave regular birth on gestation day 23.
- uterine contents were not axamined. No information available on gravid uteri weight, number of corpora lutea, uterine contents, number of implantation sites and resorptions.
- no data available on sex and litter/body weight of foetus
- no data on external alterations and skeletal and soft tisse alterations.
- information on observations of clinical signs and mortality is incomplete
- historical control data and individual data are missing. Without the inclusion of historical control data it is impossible to determine, if the findings were test item-related effects or if the findings were still in the normal range for the species/strain used.