Sodium dichromate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Reference 1

Endpoint:

fertility, other

Remarks:

male and female reproductive system

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:

This reference had reporting and experimental deficiencies as follows: test material was insufficiently described (purity and stability missing); two dose levels only for the fertility study in females; no analysis of dose levels in drinking water; water consumption data missing; mating period too short; females were not allowed to litter and no full pregnancy duration was allowed (1 week of pregnancy only); parameters missing: clinical signs, mortality, body weight (terminal weight only) food consumption, incomplete gross pathology, histopathology); individual data and historical control data missing

Qualifier:

no guideline followed

Principles of method if other than guideline:

The effects of potassium dichromate on male and female fertility were investigated in male and female Swiss mice administered this hexavalent chromium compound in drinking water. Groups of 9 - 20 males were administered 0, 1000, 2000, 4000 or 5000 mg/L potassium dichromate equivalent to doses of approximately 0, 166, 333, 666, 833 mg/kg/ bw/day (0, 60, 120, 235, 290 mg Cr(VI)/kg/day) for 12 weeks and then mated for ten days, 1 male to 2 untreated females. The exposed males were then removed and 1 week later the females were terminated. Similarly, groups of 11 - 18 females were administered 0, 2000 or 5000 mg/L potassium dichromate equivalent to doses of approximately 0, 400, 1000 mg/kg bw/day (0, 140, 350 mg Cr(VI)/kg bw/d) for 12 weeks and then mated for ten days, 3 females to 1 untreated male. One week after the removal of the males, the females were terminated. Number of pregnant females, total implantations, viable foetuses and resorptions were recorded. In addition, satellite groups of 10-13 males and 8-10 females administered 0, 2000 (males only) or 5000 mg/L potassium dichromate for 12 weeks were sacrificed at the end of the treatment. Body and reproductive organ weights were recorded in these animals.

GLP compliance:

not specified

Remarks:

not specified in the publication

Limit test:

no

Justification for study design:

not applicable

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Fluka AG, Chemische Fabrik CH-9470, Bucks

Species:

mouse

Strain:

Swiss

Details on species / strain selection:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Medical Faculty at the Jordan University of Science and Technology
- Age at study initiation (males and females): 50 days
- Diet (ad libitum)
- Water (ad libitum)

ENVIRONMENTAL CONDITIONS
- Temperature: 21 °C ± 1° C
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in drinking tap water at various concentrations.
At higher concentrations of potassium dichromate the experimental animals consumed less water per day compared to the control group.

Details on mating procedure:

EXPERIMENT 1 (male fertility):
- M/F ratio per cage: 1 treated male: 2 untreated females
- Length of cohabitation: ten days

EXPERIMENT 2 (female fertility):
- M/F ratio per cage: 1 untreated male: 3 treated females
- Length of cohabitation: ten days

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

12 weeks

Frequency of treatment:

Continuous

Details on study schedule:

not specified

Dose / conc.:

1 000 mg/L drinking water

Remarks:

Experiment 1 (male fertility)

Dose / conc.:

2 000 mg/L drinking water

Remarks:

Experiment 1 (male fertility) and Experiment 3 (satellite group; males only)

Dose / conc.:

4 000 mg/L drinking water

Remarks:

Experiment 1 (male fertility)

Dose / conc.:

5 000 mg/L drinking water

Remarks:

Experiment 1 (male fertility) and Experiment 3 (satellite group; males and females)

Dose / conc.:

2 000 mg/L drinking water

Remarks:

Experiment 2 (female fertility)

Dose / conc.:

5 000 mg/L drinking water

Remarks:

Experiment 2 (female fertility)

No. of animals per sex per dose:

Experiment 1 (male fertility): 9 - 19 males
Experiment 2 (female fertility): 11-18 females
Experiment 3 (satellite group): 13 males and 10 females

Control animals:

yes, concurrent no treatment

Details on study design:

EXPERIMENT 1 (male fertility):
After 12 week chromium exposure each male were mated with two virgin untreated females. They were left together for ten days. The chromium exposed males were then removed and 1 week later the females were sacrificed and the females were examined.

EXPERIMENT 2 (female fertility):
After 12 week chromium exposure three females were mated with one untreated males. They were left together for ten days. Chromium exposed females and control females were sacrificed 1 week after the mating period and the females were examined.

EXPERIMENT 3: (satelite group)
Satellite groups administered 0, 2000 (males only) or 5000 mg/L potassium dichromate for 12 weeks were sacrificed at the end of the treatment.

Positive control:

not specified

Parental animals: Observations and examinations:

NOTE: the following examinations apply only to experiment 3 with the satellite groups. For eperiment 1 and 2 none of the examinations were mentioned in the publication.

CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: at termination

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 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

Oestrous cyclicity (parental animals):

no specified

Sperm parameters (parental animals):

NOTE: the following examinations apply only to experiment 3 with the satellite groups

Parameters examined in all males: weights of paired testes

Litter observations:

not specified

Postmortem examinations (parental animals):

EXPERIMENT 1 (male fertility)
Females mated with test item-treated males were sacrificed one week after the mating period. The following measurements were recorded: number of pregnant females, number of viable foetuses.number of implantations, and number of resorptions or dead foetuses.

EXPERIMENT 2 (female fertility)
Test item-treated females mated with a untreated male were sacrificed one week after the mating period. The following measurements were recorded: number of pregnant females, total implantations, number of viable foetuses and number of resorptions.

EXPERIMENT 3 (satellite groups)
In males, weights of paired testes, seminal vesicles (stripped of fluid) and preputial glands were recorded. In females, weights of paired ovaries and uteri were also recorded.

Postmortem examinations (offspring):

not specified

Statistics:

Data are expressed as means ± SD. Differences between control and chromium exposed groups were analyzed using either Chi-square or Student 't' tests (Siegel, 1956)*.

*Reference:
- Siegel, S. (1956) Non-Parametric Statistics for the Behavioural Sciences. McCraw-Hill, London.

Reproductive indices:

not specified

Offspring viability indices:

no specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Experiment 3 (satellite group)
- body weight significantly decreased in 2000 mg/L and 5000 mg/L (p < 0.01) hexavalent chromium exposed males.
.
Please also refer to the field "Attached background material" below.

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

EXPERIMENT 3 (satellite group):
- testes weights were significantly increased in 2000 mg/L (p < 0.01 ), and 5000 mg/L (p < 0.05) hexavalent chromium exposed males.
Please also refer to the field "Attached background material" below.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

EXPERIMENT 2 (female fertility):
- numbers of implantations were significantly reduced in pregnant females exposed to 2000 mg/L (p < 0.01) and 5000 mg/L (p < 0.05) hexavalent chromium compounds.
- number of viable foetuses was also significantly reduced in pregnant females exposed to 2000 mg/L (p < 0.05) and 5000 mg/L (p < 0.01) of hexavalent chromium compound.
- number of pregnant female mice with resorptions was significantly increased in females exposed to 2000 mg/L (p < 0.01) and 5000 mg/L (p < 0.005) hexavalent chromium compound. The number of resorptions was high in pregnant females exposed to hexavalent chromium compounds compared to control pregnant females.
Please also refer to the field "Attached background material" below.

EXPERIMENT 1 (male fertility):
REPRODUCTIVE PERFORMANCE:
- when the adult males exposed to hexavalent chromium and unexposed control males were tested with adult untreated females for 5 minutes, males were sexually interested, as females were pursued by males, their genitalia were examined and mounting occurred.
- number of implantation sites and the number of viable foetuses were reduced significantly in females impregnated by males exposed to 2000 mg/L (p < 0.01) and 4000 mg/L (p < 0.05) hexavalent chromium compound.
- numbers of resorptions and dead foetuses were increased in females impregnated by hexavalent chromium exposed males.
Please also refer to the field "Attached background material" below.

EXPERIMENT 2 (female fertility):
- the exposure of adult female mice to hexavalent chromium compounds had no effect on female fertility (pregnancy occurred in a similar frequency control and chromium exposed females).
Please also refer to the field "Attached background material" below.

EXPERIMENT 3 (satellite group):
- exposure of female mice to 5000 mg/L hexavalent chromium had no significant effect on body weights.
.
Please also refer to the field "Attached background material" below.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/L drinking water

Sex:

male

Basis for effect level:

other: Reduced implantations. NOAEL equivalent to 166 mg/kg bw/d potassium dichromate; 60 mg/kg bw/d Cr (VI)

Dose descriptor:

LOAEL

Effect level:

2 000 mg/L drinking water

Sex:

female

Basis for effect level:

other: Reduced implantations at the lowest concentration of 2000 mg/l, equivalent to 400 mg/kg bw/d, 140 mg/kg bw.d Cr (VI)

Critical effects observed:

not specified

Remarks on result:

not measured/tested

Critical effects observed:

not specified

Reproductive effects observed:

not specified

Conclusions:

Administration of high concentrations of potassium (VI) dichromate in drinking water to male and female mice prior to mating was found to have
adverse effects on fertility (reduced numbers of implantations).

Reference 2

Endpoint:

fertility, other

Remarks:

male reproductive system

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:

This reference had reporting and experimental deficiencies as follows: test material was insufficiently described (purity and stability missing); two dose levels only instead of three dose levels as foreseen by the guideline; dose level too low; males tested only; exposure duration of 6 days is too short (guideline foresees that males should be dosed during growth and for at least one complete spermatogenic cycle (approx. 56 days in the mouse) in order to elicit any adverse effects on spermeogenesis by the test substance); no mating of treated males; parameters missing (clinical signs, mortality, body weight, food consumption, gross pathology, and histopathology (testis only investigated)); individual data and historical data missing

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 8–11 male Wistar rats were administered chromium trioxide by gavage at dose levels of 10 and 20 mg test substance/kg bw/day (equivalent to 5.2 and 10.4 mg Cr(VI)/kg bw/day, respectively) for 6 days. A control group was run concurrently. After 6 weeks, rats were sacrificed; testes and epididymis were removed and analyzed for epididymal sperm count and abnormal sperm, and testes were prepared for histological evaluations of morphological abnormalities and diameter of seminiferous tubules.

GLP compliance:

not specified

Remarks:

not specified in the publication

Limit test:

no

Justification for study design:

not applicable

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Chemical Reagent Factory (Tanjin, China)

Species:

rat

Strain:

Wistar

Details on species / strain selection:

not specified

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Experimental Animal Center of Henan Medical University (Zhenzhou, China)
- Age at study initiation: 60 days
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 1°C
- Relaive humidity: 50 ± 10 %

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION:
The test substance was given by oral feeding.

Details on mating procedure:

not specified

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

six days

Frequency of treatment:

daily

Details on study schedule:

not applicable

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Remarks:

equivalent to 5.2 mg Cr(VI)/kg bw/day

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Remarks:

equivalent to 10.4 mg Cr(VI)/kg bw/day

No. of animals per sex per dose:

8 - 11 males/group

Control animals:

yes, plain diet

Details on study design:

not sepcified

Positive control:

not specified

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: No data
BODY WEIGHT: No data

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 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE: No data

Oestrous cyclicity (parental animals):

not applicable

Sperm parameters (parental animals):

Parameters examined in all males: sperm count in epididymides, spermatozoa morphology, histological evaluation of testis (morphologic abnormalities, size dimension of seminiferou tubules)

Litter observations:

not applicable

Postmortem examinations (parental animals):

SACRIFICE
- the rats were sacrificed after 6 weeks and both testes and epididymides were removed immediately and analysed.

HISTOPATHOLOGY / ORGAN WEIGHTS
A histological evaluation of testis (morphologic abnormalities, size dimension of seminiferou tubules) was conducted.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

not applicable

Statistics:

mean and standard deviations

Reproductive indices:

not specified

Offspring viability indices:

not applicable

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

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

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 10 and 20 mg/kg/day: histopathology of testis shoed that: the diameters of the seminiferous tubules were significantly decreased after Cr(Vl) exposure. In addition, the germ cell arrangement within the seminiferious tubules was disrupted by exposure to Cr(VI)

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

- 0, 10, and 20 mg/kg/day: epididymal sperm number was 87.40 ± 3.85 x 10E6/g epididymis (N = 9) for control rats. The sperm number decreased significantly in exposed groups depending on the dose of Cr(VI) exposure. At doses of 10 and 20 mg/kg, the sperm counts were 21.40± 1.20 x10E6/g epididymis (N = 8) and 17.48 ± 1.04x10E6/g epididymis (N = 9), respectively. The differences between the exposed groups and control group were significant (P < 0.05).
- 0, 10, and 20 mg/kg/day: rates of sperm abnormality of 6.68 ± 0.32 % and 7.6 ± 0.15 % for rats exposed to 10 and 20 mg/kg/day CrO3, respectively, were significantly higher than that of 2.75 ± 0.06 % for the control group (p < 0.01).
- 10 and 20 mg/kg/day: histopathology of testis shoed that: the diameters of the seminiferous tubules were significantly decreased after Cr(Vl) exposure. In addition, the germ cell arrangement within the seminiferious tubules was disrupted by exposure to Cr(VI)

Reproductive performance:

not specified

not specified

Dose descriptor:

LOAEL

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

Dose descriptor:

LOAEL

Effect level:

5.2 mg/kg bw/day (actual dose received)

Based on:

element

Remarks:

Cr (VI)

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

Remarks on result:

other: LOAEL calculated based on test material

Critical effects observed:

not specified

Remarks on result:

not measured/tested

Critical effects observed:

not specified

Reproductive effects observed:

not specified

Conclusions:

Li, H. et al. (2001) administered chromium trioxide by gavage at dose levels of 10 and 20 mg test substance/kg bw/day (equivalent to 5.2 and 10.4 mg Cr(VI)/kg bw/day, respectively) to groups of 8–11 male Wistar rats for 6 days. A control group was run concurrently. The results showed that feeding the test substance to rats significantly reduced the epididymal sperm counts from 87.40 ± 3.85 x 10E6/g epididymis in control group to 21.40 ± 1.20 x 10E6/g epididymis at a chromium trioxide dose of 10 mg/kg body weight and to 17.48 ± 1.04x10E6/g epididymis at a chromium trioxide dose of 20 mg/kg body weight. Exposure of rats to the test substance also significantly increased the sperm abnormality from 2.75 ± 0.06% in the control group to 6.68 ± 0.32% in the exposed group at a chromium trioxide dose of 10 mg/kg body and to 7.6±0.15% at a chromium trioxide dose of 20 mg/kg body weight. In exposed rats, there was visible disruption in germ cell arrangement near the walls of the seminiferous tubules. Based on these findings a LOAEL of 10 mg/kg bw/day (equivalent to 5.2 Cr(VI)/kg bw/day) was determined for reproductive toxicity in males.

Reference 3

Endpoint:

two-generation reproductive toxicity

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:

This reference had reporting and experimental deficiencies as follows: animals were older ( approx. 10 weeks of age) as recommended by the guideline (5 -9 weeks of age) at time of testing; dosing did not start 8 weeks or at least two weeks prior to mating for males and females, respectively, but one week prior to mating; mating period was 12 weeks instead of 3 weeks as recommended by the guideline; mice were allowed to produce several litters and the last litters was used to produce the F2 generation; F1 animals were 74 ± 10 days old at the time of mating, however, the guideline foresees that the animals are at least 11 weeks old at the time of mating; food consumption was not measured during the premating period; food consumption was not measured weekly as foreseen by the guideline; F2 pups were sacrificed on postnatal day 1, whereas the guideline forsees that the F2 pups are sacrificed at weaning; duration of gestation was not recorded; presence of gross lesions in pups were not investigated

Qualifier:

no guideline followed

Principles of method if other than guideline:

The reproductive toxicity of potassium dichromate was investigated in a two-generation study in the BALB/c mouse, incorporating a continuous mating phase for the first generation. Mice (20/sex/group) were exposed to potassium dichromate in the diet for one week prior to a 12 -week continuous mating phase during which the mice were also exposed. Litters produced during this phase were sacrificed at Day 1 post partum; litters produced after this phase were raised to weaning and selected animals mated to produce litters.

GLP compliance:

yes

Limit test:

no

Justification for study design:

not applicable

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Aldrich Chemical Corp.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability of the test substance in the solvent/dispersant/vehicle/test medium: stability studies carried out on NIH-07 rodent feed containing 50 ppm potassium dichromate indicated that the formulation is best stored under refrigerator conditions (4°C). Dose formulations may bestored for 31 days with less than 9% loss in recovery during this period Storage at ambient temperatures appear to be appropriate (maximum recovery loss 7.5%) for 23 days; extending storage to 31 days at 23° - 28°C results in a.15.4% loss in recovery. Storage under freezer conditions is comparable to ambient storage although greater variability in replicate measurements (% RSD) was noted. There was no significant loss of potassium dichromate from NIH-07 feed exposed to air and light for seven days under simulated dosing conditions.

Species:

mouse

Strain:

Balb/c

Details on species / strain selection:

The BALB/c mouse was selected as the test strain since it was the species/strain selected for previous studies with potassium dichromate.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Portage, Michigan)
- Age at initiation of dosing: approx. 10 weeks
- Weight at initiation of dosing: males: 21.5 - 29.9 g; females: 15.7 - 22.2 g
- Housing: housed two per cage by sex from receipt to randomization; two per cage (one male and one female) during cohabitation; one to four per cage during holding and rearing phase (F1 generation); and housed individually at all other times. All animals were housed in polycarbonate cages 11" L x 7" W X 5"H suspended on stainless-steel racks with an Edstrom automatic watering system. Racks were equipped with filter paper liners. Polycarbonate caging contained Beta Chip® heat treated hardwood laboratory bedding.
- Diet (ad libitum): Powdered Zeigler™ NIH 07 Small Animal Feed
- Water: tap water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 23.3 °C
- Relative humidity: 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12

During the acclimation period, two male and two female mice were forwarded to Anmed Biosafe, Inc., Rockville, Maryland for determination of viral antibody titers, All sera were negative for antibodies at that time. A total of 7 male mice and 1female mouse were either found dead or moribund killed during the acclimation period. The gross necropsy did not reveal any atypical findings, although terminal body weights of these anim.als were approximately 5·12 grams less than the animals assigned to study. Two additional animals (1 male and 1 female) were submitted to Amned .Biosafe, Inc. for a core diagnostic screen and comprehensive mouse virus serology panel. The animals were determined to be free of all primary and opportunistic murine pathogens in the test battery. The mortality was most likely a result of the animaIs inability to adjust to the automatic drinking water system.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): dietary formulations of potassium dichromate and NlH-07 feed at concentrations of 100, 200, and 400 ppm were prepared for two to three weeks of use.

- Mixing appropriate amounts with NIH-07: the appropriate quantity of NIH-07 diet was accurately weighed into containers for each dose level. The appropriate quantity of test article was accurately weighed into a beaker for each dose level. A dietary premix was prepared for each dose level by adding an equal amount of the preweighed feed to the beaker containing the weighed test article and mixing with stainless steel spatula. The feed/test article was poured into a mortar and mixed with a pestle until homogeneous. The feed/test article was poured into a glass 2 L beaker and the mortar and beaker were rinsed three times with the preweighed feed. Each rinse was poured into the 2 L beaker. The feed was mixed with a spatula until homogeneous. The feed was poured in three increments that approximately doubled the amount of the feed/test article into the 2 L beaker while mixing·each increment with a spatula until homogeneous.
The dietary premix and appropriate quantity of preweighed feed were poured into the right and left ports of the Patterson·Kelly blender. 'The premix beaker was rinsed three times with preweighed feed and the remaining preweighed feed was poured into the Patterson-Kelly blender. The dietary formulation was blended for at least 90 seconds per kilogram of formulation; the intensifier bar was operating during the beginning and ending intervals of blending.

- Storage temperature of food: dietary formulations were stored refrigerated and protected from light and dispensed into glass jars with stainless steel followers and lids.


Animals were supplied with fresh feed at least once a week. For the F1 generation, animals, exposure to potassium dichromate occurred prior to actual feed consumption through the gametes, in utero, and during lactation,

Details on mating procedure:

P0 generation:
- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: 12 weeks

P1 generation:
- Length of cohabitation: breeding pairs were cohabitated until either sperm-positive, a copulatory plug was observed, or after 7 days, whichever came first.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dietary samples were collected upon completion of mixing from each dose level of every formulation, and stored in the freezer. When homogeneity samples were required, two 150 gsamples were collected from each the right, left, and bottom ports for the low and high-dose groups; two 150 g samples were collected for the other dose groups, When homogeneity samples were not required, two 150 g samples were collected for each dose level. The concentration of potassium dichromate in the feed was verified prior to the initiation of the study (P generation), half-way through treating the P generation, at the end of treating P generation, and at the start of investigating the F1 generation. Samples selected for dose concentration analysis were forwarded with a 5 g sample of bulk test article on dry ice to the testing laboratory.

Samples were analyzed by graphite furnace flame atomic absorption spectroscopy . An aliquot of each level of dose formulation ,and of the control vehicle were analyzed, Homogeneity analyses were conducted al the 100 and 400 ppm dose levels. Analysis indicated that the samples were within 87.5 - 99.6% of the target concentration and within 10 % of each other. All dose formulations analysed were within 87.5 - 130% of the nominal concentration.

Duration of treatment / exposure:

- P0 generation: 16 weeks (1 week prior to mating as well as mating period up to postnatal day 21)
- P1 generation: 13 to 16 weeks

Frequency of treatment:

ad libitum

Details on study schedule:

- Selection of parents from F1 generation when pups were 21 days of age.
On postnatal day 21, the F1 pups were separated from the P0 dams. Two male and two female weanlings were scheduled to be randomly selected from each litter. When 2 males and 2 females were not available from a litter, additional pups were selected from another litter in the same dose group on postnatal day 21. Depending on litter size, one to five males and females were selected from each litter. Animals not selected on postnatal day 21 were euthanized and discarded without necropsy. Animals not selected for mating on postnatal day 74 ± 10 were euthanized and discarded without necropsy.

Approximately one week before postnatal day 74 ± 10, at least one male and one female from each·litter were selected to obtain 20 breeding pair (P1 generation). Sibling matings were avoided. Breeding pairs were cohabitated on postnatal day 74 ±10 (please also refer to the field "Details on mating procedures" above).

Dose / conc.:

100 ppm (nominal)

Remarks:

Actual dose received:
P0 generation: 19.4 mg/kg/day (calculated based on food consumption)
P1 generation: 22.4 mg/kg/day (calculated based on food consumption)

Dose / conc.:

200 ppm (nominal)

Remarks:

Actual dose received:
P0 generation: 38.6 mg/kg/day (calculated based on food consumption)
P1 generation: 45.5 mg/kg/day (calculated based on food consumption)

Dose / conc.:

400 ppm (nominal)

Remarks:

Actual dose received:
P0 generation: 85.7 mg/kg/day (calculated based on food consumption)
P1 generation: 104.9 mg/kg/day (calculated based on food consumption)

No. of animals per sex per dose:

20 animals/sex/grup

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on slightly decreased body weights and increased feed consumption noted at 400 ppm during a previously conducted 9-week mouse study, dose levels for the continuous breeding phase of this study were set at 100, 200, and 400 ppm.

SCHEDULE OF INVESTIGATION.
The majority of dams produced three litters during continuous breeding and all litters produced during the continuous breeding phase were evaluated on postnatal day 1 and then sacrificed and discarded without necropsy. The fourth/final litter (F1 generation) was used for generating the F2 generation. However, eleven P0 generation dams produced four litters during continuous breeding and a fifth litter after continuous breeding. Pups from the fifth litter were used for generating the F2 generation. Overall, the following number of litters were produced by the dams and each tme the final litter is used for further invesigations: one dam had two litters; 11 dams had 3 litters; 44 dams had 4 litters, 11 dams had 5 litters.

At the end of the 12 week continuous breeding phase, the pairs were separated and exposure continued. Litters born (F1) after the continuous breeding phase (final litter) were reared by the dam until weaning on postnatal day 21 (please also refer to the field "Details on study schedule" above).

Positive control:

not specified

Parental animals: Observations and examinations:

1) P0 GENERATION:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: mortality and signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: study weeks 1, 5, 10 and 14 as well as on postnatal days 1, 4, 7, 14, and 21
- terminal body weights were taken

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- food consumption was measured at study weeks 1, 5, 10 and 14 (males only)
- feed consumption measurements for lactating dams were obtained on postnatal days 1, 4, 7, 11 14, 18. and 21.

WATER CONSUMPTION AND COMPOUND INTAKE: No data

2) P1 GENERATION:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: mortality and signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: postnatal day 21 and at study weeks 2 and 4.
- terminal body weights were taken

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- food consumption was measured at study weeks 2 and 4 of the P1 fertility assessment.

WATER CONSUMPTION AND COMPOUND INTAKE: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: Yes, CO2/O2 anesthesia
- Animals fasted: Not specified
- How many animals: blood was collected from the first 5 P1 animals/sex/group immediately prior to scheduled necropsy each day for hematology (total of 10 animals/sex/group).
- Parameters checked: leukocyte count, haemoglobin, haematocrit, platelet count, erythrocyte count, mean corpuscular haemoglobin concentration, mean corpuscular volume, mean corpusclar haemoglobin, and mean platelet volume
in addition, blood smears were prepared. The blood smears from all P1 animals bled at necropsy were evaluated for erythrocyte morphology.

FEMORAL BONE MARROW.
- femoral bone marrow smears were prepared.

Oestrous cyclicity (parental animals):

P1 generation:
- Vaginal smears were obtained from all females once daily for 12 days beginning four days after the delivery of the last possible litter.

Sperm parameters (parental animals):

Parameters examined in P0 and P1 male parental generations:
For the following organs the weight was determined: right testis, right cauda epididymis, and right epididymis.

For all necropsied males (P0 and P1 generation), sperm suspensions were prepared using the right vas deferens and were video taped using a computer-assisted sperm motion analysis system. The video tapes for males in the control and high-dose groups were analyzed. ln addition to the computer-assisted sperm analysis, a visual estimation of sperm motility was conducted for the first 10 surviving control P0 males and the first 5 surviving treated P0 males per group to aid in computer-assisted sperm analysis validation for BALB/c mice. Epididymal sperm density was determined and sperm smears were prepared for all necropsied males (P0 and P1 generation) from sperm suspensions prepared using the right cauda epididymis. Sperm smears from control and high-dose males were evaluated for sperm morphology. At necropsy, the right testis from all necropsied males (P0 and P1 generation) was frozen and testicular spermatid head counts were determined.

Litter observations:

1) F1 generation:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- total number of pups, number of live and dead pups, number of male and female pups and total pup weight of each sex were obtained on postnatal days 1, 4, 7, 14, and 21.
- offspring were weighed following delivery

2) F2 generation:
PARAMETERS EXAMINED
The following parameters were examined in F2 offspring:
- total number of pups, number of live and dead pups, number of male and female pups and total pup weight of each sex were obtained on postnatal day 1.
- offspring were weighed following delivery

Postmortem examinations (parental animals):

1) P0 GENERATION:
SACRIFICE:
- after weaning of the offspring of the final litter all animals were sacrificed.

2) P0 and P1 GENERATIONS:
GROSS NECROPSY
A gross necropsy was performed on all of the control animals and the first surviving 10 per sex from each of the treated groups. Necropsies included examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed: liver, kidneys; right cauda epididymis, right epididymis, prostate (ventral), seminal vesicles with coagulating glands, right testis, and ovaries. The liver (excluding the right lateral lobe), right kidney, prostate,seminal vesicles with coagulating glands, uterus, cervix, vagina, stomach, duodenum, femur wth bone marrow (P1 generaion only) and gross lesions were preserved in 10% neutral-buffered formalin for possible histopathological examination. The ovaries were preserved in Bouin's for 24 - 48 hours and then transferred to 70 % ethanol. The epididymis from all necropsied males were fixed in 4% paraformaldehyde/3% glutaraldehyde for no longer than 72 hours and then transferred to phosphate buffered saline.

The liver and kidneys from the first 5 necropsied mice/sex/group (P0 generation) or from 5 necropsied male mice/group and 10 necropsied female mice/group (P1 generation) were selected to be microscopically examined because they were considered target organs. The livers and kidneys were processed through paraffin, sectioned at 5-6 microns, stained with hematoxylin and eosin and evaluated.

Postmortem examinations (offspring):

SACRIFICE
- F2 generation: all litters were sacrificed on postnatal day 1.

Statistics:

Please refer to the field "Any other information on materials and methods incl. tables" below.

Reproductive indices:

Pregnancy index: number delivering/number cohabited
Mating index
Fertility index

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- 100 and 200 ppm: mortality was observed in eight animals (four 100 ppm females, one 200 ppm male, and three 200 ppm females). One 200 ppm female was killed moribund and gross lesions indicative of mastitis were noted at necropsy. The cause of death could not be determined for the rest of the animals but since the mortality was observed in only the low- and mid-dose groups, the mortality was apparently not treament 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:

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:

no effects observed

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:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS:
- 100, 200, and 400 ppm: incidental clinical signs noted during the study included abrasions, swellings, paraphimosis, ulcerations, tremors, hunched posture, languid behaviour, limb paralysis, hypothermia, rapid respiration, rough haircoat, exophthalmos, opacity, vaginal discharge, thinness, tissue masses, an urine stains. The incidence of these observations was low (less than 11 % per group) and no dose-related differences were observed. Alopecia was observed more frequently (5 - 30 % per group), however, because alopecia is common in mice during cohabitation and since there was no dose response, it is not considered related to treatment.

BODY WEIGHT AND WEIGHT CHANGES:
- 100, 200, and 400 ppm: sire weights, obtained on delivery day for corresponding dam, were comparable among dose groups. Mean dam weights at delivery were slightly but not always significantly decreased in the 200 and 400 ppm groups. Mean dam weights were decreased by 5%for the 200 ppm females during litter 1, and by 5 - 7 % for the 400 ppm females during litters 1-3, compared to the controls. Body weights of the P0 males and females, obtained at, Study Weeks 1,· 5, 10, and 14, were not significantly different between the control and treated groups. However, the mean body weight of the 200 and 400 ppm females were decreased by 10 and 9%, respectively, compared to control values during Study Week 14.

- 100, 200, and 400 ppm: during lactation of the final litter, the mean dam weights were decreased by 6 % for the 200 ppm females on postnatal day 14 and by 5, 7, and 11 % for the 400 ppm females on postnatal days 1, 4, and 14, respectively. Mean feed consumption data (relative and absolute) for lactating dams at the final litter on postnatal days 4, 7, 14, and 21 were generally comparable among dose groups. The mean feed consumption (relative and absolute) for the 100 ppm females was decreased by 14 - 16 % postnatal day 4. The mean calculated doses were 32.8, 69.0, and 143.1 mg/kg/day for the dams during the final litter in the 100, 200, and 400 ppm dose grous, respectively.

- 100, 200, and 400 ppm: male terminal body weights were comparable among dose groups. Mean terminal body weigts of the 400 ppm P0 females were decreased by 7 % compared to the controls.

FOOD CONSUMPTION AND COMPOUND INTAKE:
1) Mean relative feed consumption:
-100, 200, and 400 ppm: mean relative feed consumption was increased by 25, 22, and 27 % in the 100, 200, and 400 ppm males, respectively, and by 27% in the 400 ppm females, respectively, during the first week of the study (week 1). The mean relative feed consumption was comparable among these groups during study week 5, 10, and 14 (males only).

2) Mean feed consumption:
- 100, 200, and 400 ppm: mean feed consumption was increased by 23, 19, and 27 % in the 100, 200, and 400 ppm males and by 24 % in the 400 ppm females, respectively, during the first week of the study (week 1). No treatment-related differences were observed in mean feed consumption values during study weeks 5 and 10 for the cohabited males and females. However, the mean feed consumption was decreased by 8 % for the 200 ppm males and females during study week 10. Mean feed consumption values were comparable among the male dose groups during study week 14. The mean calculated doses during the study were 19.4, 38.6, and 85.7 mg/kg/day for the P0 males and females in the 100, 200, and 400 ppm dose groups, respectively.

ORGAN WEIGHT FINDINGS:
- 400 ppm: mean absolute liver weights were decreased by 17 and 12 % in the 400 ppm males and females, respectively, when compared to controls.
- 100, 200, and 400 ppm: no differences were observed in any of the other absolute organ weights (kidneys, ovaries, right causa epididymis, right epididymis, right testis, prostate, or seminal vesicles with coagulating glands). All of the relative organ weights were comparable among the dose groups for both sexes.

GROSS PATHOLOGICAL FINDINGS:
- 100, 200, and 400 ppm: no treatment-related gross lesions were observed in the P0 males and females. Incidental gross lesions noted in the P0 animals included ovarian cysts, nodules on the uterus, tan liver, and focus on the liver. These lesions were not considered treatment related because no dose response was observed.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC
- 100, 200, and 400 ppm: no treatment-related microscopic lesions were obsered in the livers or kidneys of P0 males and females. Inidental microscopic lesions included hepatocyte cytoplasmic vacuolization, acute inflammation of the liver, individual cell necrosis of hepatocytes, necrosis of the liver, renal tubule degeneration and regeneration, and perivascular lymphocyic infiltrate of the kidneys. These lesions were not considered treatment-related due to a lack of dose response.

REPRODUCTIVE FUNCTION: SPERM MEASURES
- 400 ppm: computer-assisted sperm analysis from vas deferens samples revealed no differences between the control and the high-dose group in any of the parameters (total cells analysed, sperm motility, velocity, linearity, ALH max, ALH mean, beat/cross frequency, average radius, circular cells, circular over all cells, and circular over motile cells). The mean percent abnormal sperm of the high dose group was comparable to the control group.
- 100, 200, and 400 ppm: the mean epididymal sperm density and the total number of spermatids per testis were comparable among dose groups. The mean number of spermaids per mg testis was increased by 22 % in te 200 ppm group only. The lack of a relationship to dose suggests that this was due to normal biological variability, and is not considered treatment-related.

REPRODUCTIVE PERFORMANCE:
- 100, 200, and 400 ppm: the pregnancy index was comparable among dose groups. The pregnancy index in the control and three treatment groups varied between 65 and 100% for the first four litters, all breeding pairs delivered at least two litters. The pregnancy index declined with increasing litter number for both the control· and treated dose groups. Furthermore, no treatment-related differences were observed in the mean average litters per pair.
- 100, 200, and 400 ppm: the mean cumulative days to liner was not affected by potassium dichromate. There was no difference between the treatment groups and the contol group.

Dose descriptor:

NOAEL

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:

mortality observed, non-treatment-related

Description (incidence):

- 100, 200, and 400 ppm: mortality was observed in twenty-nine animals during holding and rearing period (three males and five females in the control group, four males and six females in the low-dose group, two females in the mid dose group, and three males and six females in the high dose group). Eighteen of these animals were either found dead or killed moribund shortly after weaning (postnatal day 22 - 29). The cause of death for these 18 animals was most liekly due to weanlings being separated from the mothers at postnatal day 21. The weanlings weigted less than 10 grams at postnatal day 21 and between 17 - 23 grams at postnatal day 74 ± 10. If the pups had remained with the dam until postnatal day 28, survival would most likely have been considerably greater. The cause of death for the remaining 11 animals could not be determined. After cohabitation of the animals, mortality was observed in one control and one high dose male the cause of death could not be determined.

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:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

CLINICAL SIGNS.
- 100, 200, and 400 ppm: incidental clinical signs noted during the study included paraphimosis, hunched posture, languid behaviour, hypothermia, and thinness. These observations were mainly for one control male which was killed in extremis on postnatal day 84. The incidence of these observations was low (5 % per group) and no dose-relaed differences wer observed.

BODY WEIGHT AND WEIGHT CHANGES:
- 100, 200, and 400 ppm: no differences were observed in the mean dam or sire weights obtained following delivery.
- 100, 200, and 400 ppm: adult body weights of the P1 400 ppm males were decreased by 6 - 7 % during weeks 2 and 4 when compared to controls. Adult body weights of the P1 females were comparable among dose groups.
- 100, 200, and 400 ppm: terminal body weights of the P1 males and females were comparable among dose groups.

FOOD CONSUMPTION.
- 100, 200, and 400 ppm: during week 4, mean feed comsumption relative to body weight was increased by 41, 29, and 40 % for the 100, 200, and 400 ppm males, respectively, compared to control values. Mean relative feed consumption values were increased by 16 and 27 % for the 200 and 400 ppm females during week 2. Mean feed consumption values were increased by 35 and 30 % for the 100 and 400 ppm males and by 18 and 25 % for the 200 and 400 ppm females, respectively, during week 4 compared to control values. The mean calculated doses were 22.4, 45.5, and 104.9 mg/kg/day for the P1 males and females in the 100, 200, and 400 ppm dose groups, respectively.

HAEMATOLOGY:
- 100, 200, and 400 ppm: the mean corpuscular volume was decreased by 3 % in the 200 and 400 ppm males and by 2, 3, and 4 % in the 100, 200, and 400 ppm females, respectively, compared to controls. The mean corpuscular haemoglobin was decreased by 3 % in the 400 ppm males. Mean haemoglobin was decreased by 5 % n the 400 ppm females compared to controls. The remaining haematology parameters (white blood cells, red blood cells haematocrit, MCHC, platelet volume) were comparable among dose groups. No changes were observed in erythrocyte morphlogy.

ORGAN WEIGHTS:
- 100, 200, and 400 ppm: no differences were observed in any of the absolute organ weights. Only one incidental change was noted in the relative organ weights: the mean kidney-to-body-weight ratio was increased by 5 % in the 200 ppm females.

GROSS PATHOLOGY:
- 100, 200, and 400 ppm: only one gross lesions was observed at necropsy. One control male displayed liver foci.

- HISTOPATHOLOGICAL FINDINGS:
- 100, 200, and 400 ppm: no treatment-related microscopic lesions were observed in the livers or kidneys of the P1 males and females. Incidental microscopic lesions included hepatocyte cytoplasmic vacuolization, individual cell necrosis of hepatocytes, necrosis of the live, and renal tubule degeneration, regeneration and dilatation. These lesions were not considered treatment-related due to lack of a relationship to dose.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

REPRODUCTIVE FUNCTION: OESTRUS CYCLE:
- 100, 200, and 400 ppm: evaluation of the vaginal smears reealed no differences between the groups in the amount of time spent in the different oestrous stages, cycle length, number of cycles, number of cycling females, or number of females wit regular cycles.

REPRODUCTIVE FUNCTION: SPERM MEASURES
- 100, 200, and 400 ppm: evaluation of the computer-assisted sperm motion parameters (total cells analysed, sperm motility, velocity, linearity, ALH max, ALH mean, beat/cross frequency, average radius, circular cells, circular over all cells, and circular over motile cells) revealed no differences between the control and high-dose group. No significant differences were observed in the mean epididymal sperm density, percent abnormal sperm, spermatids/mg testis or the total spermatids per testis.

REPRODUCTIVE PERFORMANCE:
- 100, 200, and 400 ppm: measures of performance of second generation breeding pairs revealed no difference between dose groups in mating index, pregnancy index, fertility index, and gestation length.

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

- 100, 200, and 400 ppm: no treatment-related differences were observed in the mean number of live pups per litter, and proportion of pups born alive. The proportion of pups born alive was decreased by 3% in all treated groups compared to the controls, however, the decrease was only statistically significant in the 100 ppm group. During the lactation phase of the final litter, the pup survival of the F1 animals were generally comparable among groups.

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

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

BODY WEIGHT AND WEIGHT CHANGES:
- 100, 200, and 400 ppm: no treatment-related differences were observed in the absolute live pup weight or adjusted live pup weight.
- 100, 200, and 400 ppm: during the lactation phase of the final litter, the mean average pup weights were generally comparable among groups. Mean average pup weights of the 400 ppm F1 males and females were slightly (9 - 15 %) less than controls on postnatal day 14 and 21; however, these decreases did not reach statistical significance. On postnatal day 21, the mean adolescent body weigs were decreased by 16 % in the 400 ppm F1 males compared to controls. By postnatal day 74 ± 10, the mean body weights of the 400 ppm F1 males and females were decreased by 9 % when compared to te controls. The mean body weights of the 200 ppm females were creased by 4 % when compared to controls.

SEX RATIO:
- 100, 200, and 400 ppm: no treatment-related differences were observed in the sex ratio (number of males/total number of pups).

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

- 100, 200, and 400 ppm: no difference between dose groups in number of live pups per litter and proportion of pups born alive.

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

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

BODY WEIGHT AND WEIGHT CHANGES:
- 100, 200, and 400 ppm: no difference between dose groups in adjusted live pup weight. The live female pup weight was decreased by 11 % in the 400 ppm group; no differences were observed the live pup weight for males or both sexes combined.

SEX RATIO:
- 100, 200, and 400 ppm: no difference between dose groups in sex ratios of pups.

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

Reproductive effects observed:

not specified

Conclusions:

The reproductive toxicity of potassium dichromate was investigated in a two-generation study in the BALB/c mouse, incorporating a continuous mating phase for the first generation. Mice (20/sex/group) were exposed to potassium dichromate in the diet for one week prior to a 12 -week continuous mating phase during which the mice were also exposed. Litters produced during this phase were sacrificed at Day 1 post partum; litters produced after this phase were raised to weaning and selected animals mated to produce litters. The results showed no evidence of any reproductive toxicity was seen under the conditions of this study, at dose levels of up to 400 ppm (equivalent to 85.7 mg potassium dichromate/kg bw/day or 30 mg Cr/kg bw/day).

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A number of studies performed with potassium dichromate are available. Once systemically absorbed, the toxicokinetics and toxicodynamics of the water-soluble Cr (VI) salts are essentially identical, therefore the results of these studies are relevant and can be extrapolated to all members of this group. In a single generation fertility study in the mouse using administration of potassium dichromate in drinking water, effects on fertility resulting from the exposure of males and females were indicated by the reduction in the numbers of implantations; findings were apparent following exposure of females to the lowest dose level equivalent to 140 mg/kg bw/d Cr (VI). Changes in reproductive organ weights were also seen in this study but are not considered to be of clear toxicological significance (Elbetieha & Al-Hamood, 1997) as they are associated with bodyweight changes and do not have any pathological correlates. In contrast, no evidence of reproductive toxicity was seen in a NTP continuous breeding study (two generation) in the mouse using potassium dichromate at dose levels of up to 40 mg/kg bw/d Cr (VI).

In the study by Li et al.(2001), groups of 8–11 male Wistar rats (60 days old) were administered Cr(VI) trioxide by gavage at doses of 0, 10, or 20 mg Cr(VI) oxide/kg bw/d (0, 5.2, or 10.4 mg Cr(VI)/kg bw/d) for 6 days. During necropsy, testes and epididymis were removed and analyzed for epididymal sperm count and abnormal sperm; and testes were prepared (fixed in formaldehyde, embedded in paraffin, sliced, and stained with H&E) for histological evaluations of morphological abnormalities and diameter of seminiferous tubules. Epididymal sperm counts were significantly (p < 0.05) decreased by 76 and 80%, and the percentage of abnormal sperm was significantly (p < 0.01) increased by 143 and 176% in the 5.2 and 10.4 mg Cr(VI)/kg bw/d groups, respectively. Treatment-related histopathological findings included decreased diameter of seminiferous tubules and disruption of germ cell arrangement within seminiferous tubules in both treatment groups. Based on decreased sperm counts and histopathological changes to the testes, 5.2 mg Cr(VI)/kg bw/d was identified as a LOAEL for male rats exposed to gavage doses of Cr(VI) trioxide for 6 days.

 

A number of published developmental toxicity studies performed in mice with potassium dichromate are available. Mice were exposed either throughout gestation (Trivedi et al, 1989), during organogenesis (Junaid et al, 1996a) or prior to mating (Junaid et al 1996b). No evidence of teratogenicity was seen in any study, however adverse effects on fertility (reduced corpora lutea, reduced pre-implantation loss) were seen. Foetotoxicity (post-implantation loss, resorptions) and developmental toxicity (reduced skeletal ossification and subcutaneous haemorrhage) were seen consistently; findings were apparent at dose levels of 20 mg/kg bw/d Cr (VI) and above. Information on effects of Cr (VI) on the testes is available from repeated oral dose studies. In the rat, testicular degeneration was observed at a dose level (40 mg/kg bw/d (14 mg Cr(VI)/kg bw/d) which caused a large decrease in body weight gain following gavage administration of sodium dichromate for 90 days. A NOAEL of 20 mg/kg bw/d (7 mg Cr(VI)/kg bw/d) was determined for effects on the testis.

In the study by Elsaieed and Nada (2002), effects of gestational exposure to Cr(VI) were investigated in Wistar rats. Groups of 10 pregnant rats (mean initial body weight of 170 g) were administered drinking water containing 0 or 50 mg Cr(VI)/L as potassium dichromate on days 6 to 15 of gestation. During the exposure period, dams were evaluated for clinical signs of toxicity, body weights, and food and drinking water consumption. One day before delivery, rats were sacrificed and the following were evaluated: numbers of corpora lutea, pre- and post-implantation losses, resorptions, and live and dead fetuses; fetal weight; and visceral and skeletal anomalies.

No mortalities or clinical signs of toxicity were observed. Elsaieed and Nada (2002) stated that food and drinking water consumption was comparable between control and treatment groups, although data were not reported. Gestational weight gain was significantly (40%) less in treated dams, compared with controls (p < 0.05). Based on an average gestational body weight of 177 g (average calculated using body weights at mating and at the end of gestation) and the allometric equation for drinking water consumption for laboratory mammals (0.10 × body weight0.7377; U.S. EPA, 1988), a daily dose of 7.9 mg Cr(VI)/kg bw/d was estimated. In this study, treatment of rats with Cr(VI) resulted in significant (p < 0.05) increases in post-implantation loss/litter (1.5 vs. 0), resorptions/litter (1.2 vs. 0), and dead foetuses/litter (1.2 vs. 0) and decreases in live foetuses/litter (1.5 vs. 6.8 in control) and foetal weight (33% decrease). In the exposed group, increased litters with foetal anomalies were observed including visceral (renal pelvis dilation: 2.1/litter) and skeletal (incomplete skull ossification: 1.0/litter) changes; no control foetuses showed these changes.The results of this study showed that exposure of pregnant Wistar rats to drinking water containing 50 mg Cr(VI)/L as potassium dichromate (approximately 7.9 mg Cr(VI)/kg bw/d) on days 6–15 of gestation produced adverse effects on reproductive outcome and foetal development. Thus a LOAEL of 7.9 mg Cr(VI)/kg bw/d was identified from this study.

 

Other studies found no effects on the testis, following administration of potassium dichromate by the dietary route for 9 weeks. The highest dose levels in these studies were 24 mg/kg bw/d (8 mg Cr(VI)/kg bw/d) in the rat and 92 mg/kg bw/d (32 mg Cr(VI)/kg bw/d) in the mouse (EU RAR, 2005). Summary and discussion of the available reproductive toxicity studies from the EU RAR (2005) There are three animal studies available which focus on fertility. Adverse effects were produced in mice receiving potassium dichromate for 12 weeks in drinking water at 333 mg/kg bw/d (120 mg Cr(VI)/kg bw/d) and 400 mg/kg be/d (140 mg Cr(VI)/kg bw/d) and above in males and females respectively. A NOAEL of 166 mg/kg bw/d (60 mg Cr(VI)/kg bw/d) was identified in males, but no NOAEL was found for females as 400 mg/kg bw/d was the lowest dose level tested. An increase in resorptions following treatment of males and a decrease in implantations in treated females were among the findings in this study. In another study, pregestational oral administration of potassium dichromate in drinking water to female mice produced adverse effects on fertility (reduced number of corpora lutea and increased pre-implantation loss) at 500 ppm (119 mg/kg bw/d (40 mg Cr(VI)/kg bw/d)) and above. NOAEL values of 119 mg/kg bw/d (40 mg Cr(VI)/kg bw/d) and 63 mg/kg bw/d (20 mg Cr(VI)/kg bw/d) can be identified from this study for maternal toxicity and fertility effects respectively. In a third study, also in the mouse, at 86 mg/kg bw/d (30 mg Cr(VI)/kg bw/d), the highest dose level tested, there were no effects of treatment on fertility parameters. Fetotoxicity, including post-implantation losses, has been observed in the mouse following administration of potassium dichromate in drinking water during gestation (days 0-19). Significant developmental effects occurred at the lowest dose level tested, 60 mg/kg bw/d (20 mg Cr(VI)/kg bw/d) in the absence of maternal toxicity. Therefore no developmental NOAEL was determined. Qualitatively similar results were obtained in another study in which (350 mg/kg) potassium dichromate (125 mg Cr(VI)/kg) was administered for a shorter period, on days 6-14 of gestation. In a pregestational study in female mice, fetotoxic effects were seen starting from the lowest dose level tested, 250 ppm (63 mg/kg bw/d (22.1 mg Cr(VI)/kg bw/d)) potassium dichromate. Significant levels of total chromium were found in treated animals at sacrifice. No NOAEL could be identified for the developmental effects, which included post-implantation losses. These fetal effects may possibly be explained by the presence of chromium in the dams after the end of treatment. Overall, highly water soluble chromium (VI) compounds should be considered to be developmental toxicants in the mouse.

These findings can be regarded as relevant to humans. No reproductive toxicity studies are available using the inhalation or dermal routes of exposure.

Human data relating to effects on reproduction are limited to poorly reported studies of female workers from which no conclusions can be drawn.

Effects on developmental toxicity

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

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:

This reference had reporting and experimental deficiencies as follows: test item was insufficiently characterised (purity and stability missing); number of pregnant female per group is too low (n = 10; guideline foresees approx. 20 female animals); dosing was not conducted up to sacrifice of the dams (dosing: gestation day 6 to gestation day 15); one dose level only (guideline foresees three dose levels); dose levels in drinking water were not analysed (drinking water study); foetal sex was not determined; 1/3 and 2/3 of the foetuses were examined for visceral and skeletal malformation, respectively, whereas the guideline foresees an examination of 1/2 of each litter for visceral and skeletal examination each; body weight was measured weekly (guideline foresees another schedule); no gross pathology of dams; no uteri weight incl cervix; no difference was made between early and late resorptions; individual and historical control data were missing

Qualifier:

no guideline followed

Principles of method if other than guideline:

A group of 10 pregnant female Wistar rats was administered potassium chromate via drinking water daily at a dose level of 50 ppm (equivalent to 5 mg/kg or 1.35 mg Cr/kg) during gestation days 6 - 15. A control group was run concurrently (10 pregnant female rats). The pregnant rats were sacrificed on gestation day 20 and the following parameters were investigated: clinical signs, body weight, food consumption, water consumption, chromium levels in blood, ovarian and uterine contents (number of corpora lutea, implantation sites, resorptions, dead foetuses, and live foetuses). Furthermore, the following parameters were investigated in the foetuses: foetal weight (dead and alive), external abnormalities (dead and alive foetus), skeletal abnormalities (dead and alive foetus), and visceral abnormalities (dead and alive foetuses). Lastly, the chromium level in the placenta and the foetuses was determined by atomic absorption spectroscopy.

GLP compliance:

not specified

Remarks:

not specified in the publication

Limit test:

no

Specific details on test material used for the study:

not specified

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age: 3 months
- Weight: 170 ± 27 g
- Housing: housed individually in separate cages
- Diet (ad libitum)
- Water (ad libitum)

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was contained in drinking water supplied to the test animals.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- Proof of pregnancy: sperm in vagina referred to as day 0 of pregnancy

Duration of treatment / exposure:

gestation day 6 to gestation day 15

Frequency of treatment:

daily

Duration of test:

20 days

Dose / conc.:

50 ppm

Remarks:

equivalent to 5 mg/kg (calculated by authors based on water consumption)
equivalent to 1.35 mg Cr/kg (calculated based on 5 mg/kg of test item)

No. of animals per sex per dose:

10 pregnant females

Control animals:

yes

Details on study design:

- Dose selection rationale: chromium dosage was seleted on the basis of a preliminary study, that 100 ppm caused complete pre-implantation loss and on the fact that this dose was approximately equivalent to the 1/30 of the oral LD50 for female rats (Hertel 1982)*.

*Reference:
- Hertel, R. (1982) Chromium as a problem in hysiology epidemiology and biological monitoring. Staub Reinhalt Luft 42: 135 - 137.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: signs of toxicity

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: every week

FOOD CONSUMPTIONE: Yes
- Food consumption was recorded (time schedule of determination not stated).

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Water consumption was recorded (time schedule of determination not stated).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20

CHROMIUM LEVELS IN BLOOD:
A heparinized blood sample from each female was collected (3 mL). Total chromium content of maternal blood was determined by Atomic Absorption Spectroscopy.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations/litter: Yes
- Number of resorptions/litter: Yes
- Pre- and post implantation losses per litter were recorded
- total number of dead and alive foetuses per litter were recorded.

Fetal examinations:

- External examinations: Yes (dead and alive foetuses)
- Soft tissue examinations: Yes (1/3 of both dead and alive foetuses)
- Skeletal examinations: Yes (2/3 of both dead and alive foetuses)
- Head examinations: No data

- dead and alive foetuses were weighed.
- one foetuse with its placenta was taken from each female for metal analysis. Total chromium contents of placenta and foetal tissues were determined by Atomic Absorption Spectroscopy. Chromium was measured following digestion of the samples in a hot HNO3:HCl (1:3) mixture and oxidizing the clear extract by potassium permanganate (AOAC, 1984)*.
- histopathological examination was performed on placental tissue (Drury et al. 1976)*.

*Reference:
- AOAC (1984) Officail Methods of Analysis, Metal and other Elements in Food, 14th ed. AOAC, Arlington, VA: 444.
- Drury, R. Wallington, E. Cacerson, R. (1976) Carlton's Histopathological Techniques, 4th ed. Oxford Univ. Press, London, New York, Tronto.

Statistics:

Data of foetotoxicity and chromium estimation were analysed by one way ANOVA (Gad and Weil, 1982)*.

*Reference:
- Gad, S., Weil C. (1982) Statistics of toxicologists In: Wallace Hayes (ed.) Principles and Mthods of Toxicology. RAven Press, New York.

Indices:

not specified

Historical control data:

not specified

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- significant reduction in gestational weight gain was observed for the dams of the treated group when compared with the control group (control group: 23.6 ± 1.3 g; treatment group: 14.2 ± 1.7 g; p < 0.05).

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

no effects observed

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:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

CHROMIUM LEVELS IN BLOOD:
- dams exposed to chromium showed a significant increase of chromium levels in their blood (control group: 0.013 ± 0.06; treatment group: 0.06 ± 0.008; p < 0.05).

Details on results:

CLINICAL SIGNS & MORTALITY:
- dams of all groups were active and did not show any signs of toxicity or any notable changes in their behaviour.

FOOD CONSUMPTION:
- no significant difference in food intake was recorded among control and treated group during gestation period.

WATER CONSUMPTION:
- no significant difference in water consumption were recorded among control and treated group during gestation period.

Number of abortions:

not specified

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

- dams of the treatment group had significant increases in the numbers of pre- and post-implantation losses (Pre-implantation loss/litter: control group: 0; treatment group: 2.1 ± 0.36; p < 0.05)(Post-implantation loss/litter: control group: 0; treatment group: 1.5 ± 0.34; p < 0.05).
- complete absence of implantation in uterine horns in one dam was observed, while corpora lutea were present.

Total litter losses by resorption:

not specified

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

- a significant increase in number of resorption/litter was observed in the treatment group compared to the control group (control group: 0; treatment group: 1.2 ± 0.13; p < 0.05).

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

- a significant increase in number of dead foetuses/litter was observed in the treatment group compared to the control group (control group: 0.1 ± 0.099; treatment group: 1.2 ± 0.24; p < 0.05).

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

- intrauterine hemorrhage is recorded in some females of the treatment group.

Details on maternal toxic effects:

CORPORA LUTEA:
- no significant differences in the number of corpora lutea were observed for the treatment group compared to the control.

Remarks on result:

other: see "Remarks"

Remarks:

The results showed no treatment related effects on clinical signs, food consumption, water consumption, and number of corpora lutea. However, a statistically significant reduction in gestational weight gain was observed on the dams of the treated group when compared with the control group. In addition, dams of the treatment group had statistically significant increases in the numbers of pre- and post-implantation losses as well as in number of resorption/litter and dead foetuses/litter compared to the control group. Complete absence of implantation in uterine horns in one dam was observed, while corpora lutea were present. Intrauterine hemorrhage is recorded in some females of the treatment group. Furthermore, dams exposed to chromium showed a statistically significant increase of chromium levels in their blood

Abnormalities:

not specified

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

- significant decrease in foetal weight was recorded for the treatment group compared with the control group (control group: 3.9 ± 0.42; treatment group: 2.6 ± 0.23; p < 0.05)

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

- significant decrease in number of live foetuses was recorded for the treatment group compared with the control group (control group: 6.8 ± 0.44; treatment group: 1.5 ± 0.29; p < 0.05)

Changes in sex ratio:

not specified

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not specified

External malformations:

not specified

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Skeletal examination of the obtained foetuses revealed a significant increase in the numbers of incomplete ossification of the skull bone. Overall, a significant increase in number of skeletal anomalies/litter was observed in the treatment group compared to the control group (control group: 0; treatment group: 1.0 ± 0.34; p < 0.05).

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Visceral examination of the obtained foetuses revealed a significant increase in the numbers of renal pelvis dilatation. Overall, a significant increase in number of visceral anomalies/litter was observed in the treatment group compared to the control group (control group: 0; treatment group: 2.1 ± 0.39; p < 0.05).

Other effects:

effects observed, treatment-related

Description (incidence and severity):

- chromium induced histopathological lesions in the placenta and dams exposed to chromium showed a significant increase of chromium levels in the placenta (control group: 0.04 ± 0.003; treatment group: 0.2 ± 0.019; p < 0.05). Moreover, it passed the placental barrier and accumulated in the foetal tissues (control group: 0.015 ± 0.003; treatment group: 0.13 ± 0.03; p < 0.05). However, the placental tissue had higher chromium content than the foetal tissues and foetal/placental ratio significant increased in the treatment group compared to the control group (control group: 0.37 ± 0.041; treatment group: 0.65 ± 0.072; p < 0.05)

Details on embryotoxic / teratogenic effects:

not specified

Remarks on result:

other: see "Remarks"

Remarks:

A statistically significant decrease in foetal weight and number of live foetuses was recorded for the treatment group compared with the control group. Furthermore, skeletal examination of the obtained foetuses revealed a significant increase in the numbers of incomplete ossification of the skull bone. In addition, visceral examination of the obtained foetuses revealed a significant increase in the numbers of renal pelvis dilatation. Overall, a significant increase in number of skeletal anomalies/litter and visceral anomalies/litter was observed in the treatment group compared to the control group. Lastly, chromium induced histopathological lesions in the placenta and dams exposed to chromium showed a statistically significant increase of chromium levels in the placenta. Moreover, it passed the placental barrier and accumulated in the foetal tissues. However, the placental tissue had higher chromium content than the foetal tissues and foetal/placental ratio statstically significant increased in the treatment group compared to the control group.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Elsaieed and Nada (2002) administered 50 ppm potassium chromate (equivalent to 5 mg/kg or 1.35 mg Cr/kg) via drinking water daily to a group of 10 pregnant female Wistar rats during gestation days 6 - 15. A control group was run concurrently (10 pregnant female rats). The pregnant rats were sacrificed on gestation day 20. The results showed no treatment related effects on clinical signs, food consumption, water consumption, and number of corpora lutea. However, a statistically significant reduction in gestational weight gain was observed on the dams of the treated group when compared with the control group. In addition, dams of the treatment group had statistically significant increases in the numbers of pre- and post-implantation losses as well as in number of resorption/litter and dead foetuses/litter compared to the control group. Complete absence of implantation in uterine horns in one dam was observed, while corpora lutea were present. Intrauterine hemorrhage is recorded in some females of the treatment group. Furthermore, dams exposed to chromium showed a statistically significant increase of chromium levels in their blood

Concering the foetuses, a statistically significant decrease in foetal weight and number of live foetuses was recorded for the treatment group compared with the control group. Furthermore, skeletal examination of the obtained foetuses revealed a significant increase in the numbers of incomplete ossification of the skull bone. In addition, visceral examination of the obtained foetuses revealed a significant increase in the numbers of renal pelvis dilatation. Overall, a significant increase in number of skeletal anomalies/litter and visceral anomalies/litter was observed in the treatment group compared to the control group. Lastly, chromium induced histopathological lesions in the placenta and dams exposed to chromium showed a statistically significant increase of chromium levels in the placenta. Moreover, it passed the placental barrier and accumulated in the foetal tissues. However, the placental tissue had higher chromium content than the foetal tissues and foetal/placental ratio statstically significant increased in the treatment group compared to the control group.

Reference 2

Endpoint:

developmental toxicity

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:

This reference had reporting and experimental deficiencies as follows: number of pregnant female per group is too low (n = 10; guideline foresees approx. 20 female animals); dose levels in drinking water were not analysed (drinking water study); foetal sex was not determined; 1/3 and 2/3 of the foetuses were examined for visceral and skeletal malformation, respectively, whereas the guideline foresees an examination of 1/3 to 1/2 of each litter for skeletal anomalies and the reamining foetuses for visceral anomalies; 2/3 of the foetuses were examined externally, whereas the guideline foresees that all foetueses should be examined; no gross pathology of dams; food consumption was not recorded for the dams; individual and historical control data were missing

Qualifier:

no guideline followed

Principles of method if other than guideline:

A group of 10 pregnant female Swiss mice was administered potassium dichromate via drinking water daily at dose levels of 250, 500, and 750 ppm (2, 3.75, and 5.47 mg Cr/mouse/day, respectively) during gestation days 6 - 14. A control group was run concurrently (10 pregnant female rats). The pregnant rats were sacrificed on gestation day 19 and the following parameters were investigated: clinical signs, mortality, body weight, water consumption, chromium levels in blood, ovarian and uterine contents (number of corpora lutea, resorptions, dead foetuses, and live foetuses). Furthermore, the following parameters were investigated in the foetuses: foetal weight, placental weight, crown-rump length, external abnormalities, skeletal abnormalities, and visceral abnormalities. Lastly, the chromium level in the placenta and the foetuses was determined.

GLP compliance:

not specified

Remarks:

not specified in the publication

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Ranbaxy Laboratories Ltd., India

Species:

mouse

Strain:

Swiss

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Industrial Toxicology Research Centre bred colony
- Age: adult
- Weight: 30 ± 5 g
- Housing: kept in plastic cages individually under standard animal care conditions.
- Diet (ad libitum): pellet feed (chromium level 1.45 µg/g; Lipton India Ltd.)
- Water (ad libitum)

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on exposure:

Pregnant mice were exposed to potassium dichromate in the drinking water.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused (overnight)
- M/F ratio per cage: 1 male / 1 female
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Gestation days 6 - 14

Frequency of treatment:

Continuous

Duration of test:

19 days

Dose / conc.:

250 ppm (nominal)

Remarks:

2.00 mg Cr/mouse/day (determined based on water consumption)

Dose / conc.:

500 ppm (nominal)

Remarks:

3.75 mg Cr/mouse/day (determined based on water consumption)

Dose / conc.:

750 ppm (nominal)

Remarks:

5.47 mg Cr/mouse/day (determined based on water consumption)

No. of animals per sex per dose:

10 pregnant female mice

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: the dose was selected on the basis of earlier studies conducted by the authors (Trivedi, B. et al. (1989); Junaid, M. et al. (1995;1996))*.

*References:
- Trivedi, B., Saxena, D.K., Murthy, R.C. and Chandra, S.V. (1989) Embryotoxicity and fetotoxicity of orally administered hexavalent chromium in mice. Reproduct. Toxicol. 3, 275-218.
- Junaid, M., Murthy, R.C. and Saxena, D.K. (1995) Chromium fetotoxicity in mice during late pregnancy. Vet. Hum. Toxicol. 37, 320-322.
- Junaid, M., Murthy, R.C. and Saxena, D.K. (1996) Embryo and fetotoxicity of chromium in pregestationally exposed mice. Bull Environ Contam Toxic01 (in press).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked:physical signs of toxiciy and mortality

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE: No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 19

CHROMIUM ESTIMATION:
Blood was withdrawn from the heart and chromium content was determined.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of resorptions: Yes
- Number of foetuses/litter were recorded.
- Number of live/dead foetuses
- Post-implantation loss was calculated

Fetal examinations:

- Soft tissue examinations: Yes (1/3 of foetuses/litter)
- External examinations: Yes (2/3 of foetuses/litter)
- Skeletal examinations: Yes (2/3 of foetuses/litter; same foetuses as used for external examiniation)
- Head examinations: No data

- Weight of foetuses and placentae was recorded.
- Crown-rump lngth of the foeuses was determined.
- One foetus/litter with its placenta was kept for chromium estimation.

Placenta and foetuses were washed with saline, blotted dry and weighed. They were then digested in a HNO3:HClO4 (6:1) mixture till a white residue remained. This residue was dissolved in an appropriate amount of 0.1 N HNO3 and chromium was estimated on a DC plasma emission spectrophotometer. Blank and chromium-spiked samples were run and analysed simultaneously (NIOSH (1987); Berman, E. (1980))*.

*References:
- NIOSH (1987). Manual of analytical methods, 3rd ed. US Department of Health and Human Services. Public Health service, Centre for Disease Control. National Institute of Occupational Safety and Health, Washington DC, 8005.
- Berman, E. (1980) Toxic metals and their analysis. In: L.C. Thomas (Ed.), Hyden International Topics in Science Series, Hyden, London, pp. 74.

Statistics:

Overall significance of differences in mean values between control and treatment groups was tested using two way analysis of variance (ANOVA) where each treatment was randomly assigned a block; an equal number of replicates of the treatment represent the number of blocks. Prior to the analysis, normality assumption of the data and homogeneity of variance between the experimental groups was ascertained. The means of the experimental groups and the controls and
between two treatments were compared separately using Dunett’s test (Zar, J.H. (1984))*. Significant differences in incidences of gross and skeletal abnormalities between groups the mid and hgh dose levels were tested using Fisher’s exact test as all the expected frequencies were less than five.

*Reference:
- Zar, J.H. (1984). Biostatistical Analysis. 2nd Edn., Englewood Cliffs, NJ, Prentice HalI, 194-195.

Indices:

not specified

Historical control data:

not specified

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- dams of the 500 and 750 ppm dose groups had a statistically significant reduction in gestational weight gain (14.29 ± 0.54 g and 11.79 ± 0.49, respectively; p < 0.05) compared to the control group (15.57 ± 0.20 g). Compared with the control group, the 500 and 750 ppm dose groups revealed a 8.2 % and 24 % reductions, respectively.

Please also refer to the field "Attached background material" below.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

no effects observed

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:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

CHROMIUM ESTIMATION:
Chromium level was found to be dose-dependent in the treated mice as evidenced from a significantly higher metallic level in maternal blood compared to the control group (control group: 0.033 ± 0.003 µg/mL; 250 ppm group: 0.040 ± 0.004 µg/mL; 500 ppm group: 0.045 ± 0.003 µg/mL; 750 ppm group: 0.058 ± 0.004 µg/mL; p < 0.05).

Please also refer to the field "Attached background material" below.

Details on results:

CLINICAL SIGNS:
- no notable changes in behaviour or clinical signs were observed in control or in treated dams.

MORTALITY:
- no mortality was observed during the experimental period.

WATER CONSUMPTION AND COMOUND INTAKE:
- daily water consumption was 8.8, 8.0, 7.5 and 7.3 mL/mouse/day for the dose level groups 0, 250, 500, and 750 ppm, respectively. Based on this water intake, the chromium level was 2.00, 3.75 and 5.47 mg/mouse/day for the dose level groups 250, 500, and 750 ppm, respectively.

Number of abortions:

not specified

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant higher incidences of post-implantation loss was observed in the 500 and 750 dose group compared to the control group (control group: 4.32% ± 2.34; 500 ppm group: 21.93% ± 3.96; 750 ppm group: 34.60% ± 2.54; p < 0.05).

Please also refer to the field "Attached background material" below.

Total litter losses by resorption:

not specified

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

The number of resorption sites was found to be dose-dependent. The higher dose showed a higher number of resorption sites than the lower one. All dose groups had a statistically significant increase in number of resorption sites compared to the control (control group: 0.30 ± 0.21; 250 ppm group: 1.00 ± 0.21; 500 ppm group: 1.70 ± 0.3; 750 ppm group: 2.30 ± 0.273; p < 0.05).

Please also refer to the field "Attached background material" below.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

The incidences of dead foetuses were increased in 750 ppm group compared to the other groups (control group: all live; 250 ppm group: all live; 500 ppm group: 3 dead foetuses (2 litters); 750 ppm group: 12 dead foetuses (7 litters).

Please also refer to the field "Attached background material" below.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

not specified

Details on maternal toxic effects:

not specified

Remarks on result:

other: see "Remarks"

Remarks:

The results showed no treatment-related effect on clinical signs, mortality, and water consumption. However, dams of the 500 and 750 ppm dose groups had a statistically significant reduction in gestational weight gain compared to the control group. Compared with the control group, the 500 and 750 ppm dose groups revealed a 8.2 % and 24 % reductions, respectively. Furthermore, a statistically significant higher incidences of post-implantation loss was observed in the 500 and 750 dose group compared to the control group. In addition, the number of resorption sites was found to be dose-dependent. The high dose level showed a higher number of resorption sites than the lower ones. All dose groups had a statistically significant increase in number of resorption sites compared to the control. The incidences of dead foetuses were also increased in 750 ppm group compared to the other groups. Lastly, the chromium level was found to be dose-dependent in the treated mice as evidenced from a significantly higher metallic level in maternal blood compared to the control group.

Abnormalities:

not specified

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

- foetal weight was statistically significant reduced in the mid and high dose group compared to the control group (control group: 1.31 ± 0.41g; 500 ppm group:1.14 ± 0.03; 750 ppm group:1.06 ± 0.029 g; p < 0.05), the 750 ppm group showing higher weight reduction (19%) than the 500 ppm group (13%).

Please also refer to the field "Attached background material" below.

Reduction in number of live offspring:

not specified

Changes in sex ratio:

not specified

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

- the number of foetuses per litter was statistically significant reduced in the mid and high dose groups when compared to controls (control group: 8.8 ± 0.29/litter; 500 ppm group: 7.00 ± 0.36/litter; 750 ppm group: 7.20 ± 0.24/litter; p < 0.05) and the low dose group (250 ppm group: 8.20 ± 0.20/litter; p < 0.05). However, no marked difference were observed when both were compared.

Please also refer to the field "Attached background material" below.

Changes in postnatal survival:

not specified

External malformations:

effects observed, treatment-related

Description (incidence and severity):

There were statistically significant increases of gross structural abnormalities in the high dose group, namely, drooping of the wrist (carpal flexure)(control group: 0/40 foetuses; 750 ppm group: 4/25 foetuses (16 %; p < 0.05)) and subdermal hemorrhagic patches on the thoracic and abdominal part (control group: 0/40 foetuses; 750 ppm group: 4/25 foetuses (16 %; p < 0.05)).

Please also refer to the field "Attached background material" below.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant increases in the incidences of reduced ossification in nasal, frontal, parietal, interparietal, caudal and tarsal bones was observed in the 750 ppm dosed group, whereas the 500 ppm dosed group revealed statistically significant incidences of reduced ossification in caudal bones only.
Reduced nasal ossification: control group: 0/40 foetuses; 750 ppm group: 16/25 (64 %; p < 0.05)
Reduced frontal ossification: control group: 0/40 foetuses; 750 ppm group: 10/25 (40 %; p < 0.05)
Reduced parietal ossification: control group: 0/40 foetuses; 750 ppm group: 8/25 (32 %; p < 0.05)
Reduced interparital ossification: control group: 0/40 foetuses; 750 ppm group: 10/25 (40 %; p < 0.05)
Reduced caudal ossification: control group: 1/40 foetuses (2.5 %); 250 ppm group: 3/40 foetuses (7.5 %); 500 ppm group: 14/30 foetuses (46.6 %); 750 ppm group: 21/25 (84 %; p < 0.05 for mid nd high dose group only)
Reduced tarsal ossification: control group: 0/40 foetuses; 750 ppm group: 19/25 (76 %; p < 0.05)

Please also refer to the field "Attached background material" below.

Visceral malformations:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

CHROMIUM ESTIMATION:
Chromium level was found to be dose-dependent in the treated mice as evidenced from a significantly higher metallic level in placenta and foetuses. The rate of transfer of chromium from the mother to placenta and from placenta to foetus, calculated as ratio, revealed that in the 750 ppm group the placental metal level was more than the other treated groups, which showed almost the same chromium transfer. The transfer ratio from placenta to foetus did not show any change in any of the treated groups.
Placenta:
control group: 0.07 ± 0.001 µg/g fresh weight; 250 ppm group: 0.09 ± 0.001 µg/g fresh weight; 500 ppm group: 0.12 ± 0.001 µg/g fresh weight; 750 ppm group: 0.20 ± 0.006 µg/g fresh weight; p < 0.05
Foetus:
control group: 0.042 ± 0.0007 µg/g fresh weight; 250 ppm group: 0.056 ± 0.001 µg/g fresh weight; 500 ppm group: 0.069 ± 0.009 µg/g fresh weight; 750 ppm group: 0.10 ± 0.004 µg/g fresh weight; p < 0.05

Please also refer to the field "Attached background material" below.

Details on embryotoxic / teratogenic effects:

EXTERNAL MALFORMATIONS:
- there was no significant gross structural abnormalities in the 250 ppm and 500 ppm group, except for drooping wrist (control group: 0/40 foetuses; 250 ppm group: 3/40 foetuses (7.5 %); 500 ppm group: 3/30 foetuses (10 %); not statistically significant) and subdermal hemorrhagic patches (control group: 0/40 foetuses; 500 ppm group: 3/30 foetuses (10 %); not statistically significant).

VISCERLA MALFORMATIONS
No gross visceral abnormality was seen in any of the treated groups.

Please also refer to the field "Attached background material" below.

Remarks on result:

other: see "Remarks"

Remarks:

Foetal weight was statistically significant reduced in the mid and high dose groups compared to the control group, the 750 ppm group showing higher weight reduction (19%) than the 500 ppm group (13%). Moreover, the number of foetuses per litter was statistically significant reduced in the mid and high dose groups when compared to controls and the low dose group. However, no marked difference were observed when both were compared. In addition, there were statistically significant increases of gross structural abnormalities, namely, drooping of the wrist (carpal flexure; 16 %) and subdermal hemorrhagic patches on the thoracic and abdominal part (16 %). Statistically significant increases in the incidences of reduced ossification in nasal, frontal, parietal, interparietal, caudal and tarsal bones was observed in the 750 ppm dosed group, whereas the 500 ppm dosed group revealed statistically significant incidences of reduced ossification in caudal bones only. No treatment-related effect was observed for visceral abnormalities. Lastly, chromium level was found to be dose-dependent in the treated mice as evidenced from a significantly higher metallic level in placenta and foetuses. The rate of transfer of chromium from the mother to placenta and from placenta to foetus, calculated as ratio, revealed that in the 750 ppm group the placental metal level was more than the other treated groups, which showed almost the same chromium transfer. The transfer ratio from placenta to foetus did not show any change in any of the treated groups.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Junaid, M. et al. (1996) administered 250, 500, and 750 ppm of potassium dichromate (2, 3.75, and 5.47 mg Cr/mouse/day, respectively) via drinking water daily to a group of 10 pregnant female Swiss mice during gestation days 6 - 14. A control group was run concurrently (10 pregnant female rats). The pregnant rats were sacrificed on gestation day 19. The results showed no treatment-related effect on clinical signs, mortality, and water consumption. However, dams of the 500 and 750 ppm dose groups had a statistically significant reduction in gestational weight gain compared to the control group. Compared with the control group, the 500 and 750 ppm dose groups revealed a 8.2 % and 24 % reductions, respectively. Furthermore, a statistically significant higher incidences of post-implantation loss was observed in the 500 and 750 dose group compared to the control group. In addition, the number of resorption sites was found to be dose-dependent. The high dose level showed a higher number of resorption sites than the lower ones. All dose groups had a statistically significant increase in number of resorption sites compared to the control. The incidences of dead foetuses were also increased in 750 ppm group compared to the other groups. Lastly, the chromium level was found to be dose-dependent in the treated mice as evidenced from a significantly higher metallic level in maternal blood compared to the control group.

Foetal weight was statistically significant reduced in the mid and high dose groups compared to the control group, the 750 ppm group showing higher weight reduction (19%) than the 500 ppm group (13%). Moreover, the number of foetuses per litter was statistically significant reduced in the mid and high dose groups when compared to controls and the low dose group. However, no marked difference were observed when both were compared. In addition, there were statistically significant increases of gross structural abnormalities, namely, drooping of the wrist (carpal flexure; 16 %) and subdermal hemorrhagic patches on the thoracic and abdominal part (16 %). Statistically significant increases in the incidences of reduced ossification in nasal, frontal, parietal, interparietal, caudal and tarsal bones was observed in the 750 ppm dosed group, whereas the 500 ppm dosed group revealed statistically significant incidences of reduced ossification in caudal bones only. No treatment-related effect was observed for visceral abnormalities. Lastly, chromium level was found to be dose-dependent in the treated mice as evidenced from a significantly higher metallic level in placenta and foetuses. The rate of transfer of chromium from the mother to placenta and from placenta to foetus, calculated as ratio, revealed that in the 750 ppm group the placental metal level was more than the other treated groups, which showed almost the same chromium transfer. The transfer ratio from placenta to foetus did not show any change in any of the treated groups.

Toxicity to reproduction: other studies

Additional information

A number of studies performed with potassium dichromate are available. Once systemically absorbed, the toxicokinetics and toxicodynamics of the water-soluble Cr (VI) salts are essentially identical, therefore the results of these studies are relevant and can be extrapolated to all members of this group. In a single generation fertility study in the mouse using administration of potassium dichromate in drinking water, effects on fertility resulting from the exposure of males and females were indicated by the reduction in the numbers of implantations; findings were apparent following exposure of females to the lowest dose level equivalent to 140 mg/kg bw/d Cr (VI). Changes in reproductive organ weights were also seen in this study but are not considered to be of clear toxicological significance (Elbetieha & Al-Hamood, 1997) as they are associated with bodyweight changes and do not have any pathological correlates. In contrast, no evidence of reproductive toxicity was seen in a NTP continuous breeding study (two generation) in the mouse using potassium dichromate at dose levels of up to 40 mg/kg bw/d Cr (VI).

In the study by Li et al.(2001), groups of 8–11 male Wistar rats (60 days old) were administered Cr(VI) trioxide by gavage at doses of 0, 10, or 20 mg Cr(VI) oxide/kg bw/d (0, 5.2, or 10.4 mg Cr(VI)/kg bw/d) for 6 days. During necropsy, testes and epididymis were removed and analyzed for epididymal sperm count and abnormal sperm; and testes were prepared (fixed in formaldehyde, embedded in paraffin, sliced, and stained with H&E) for histological evaluations of morphological abnormalities and diameter of seminiferous tubules. Epididymal sperm counts were significantly (p < 0.05) decreased by 76 and 80%, and the percentage of abnormal sperm was significantly (p < 0.01) increased by 143 and 176% in the 5.2 and 10.4 mg Cr(VI)/kg bw/d groups, respectively. Treatment-related histopathological findings included decreased diameter of seminiferous tubules and disruption of germ cell arrangement within seminiferous tubules in both treatment groups. Based on decreased sperm counts and histopathological changes to the testes, 5.2 mg Cr(VI)/kg bw/d was identified as a LOAEL for male rats exposed to gavage doses of Cr(VI) trioxide for 6 days.

 

A number of published developmental toxicity studies performed in mice with potassium dichromate are available. Mice were exposed either throughout gestation (Trivedi et al, 1989), during organogenesis (Junaid et al, 1996a) or prior to mating (Junaid et al 1996b). No evidence of teratogenicity was seen in any study, however adverse effects on fertility (reduced corpora lutea, reduced pre-implantation loss) were seen. Foetotoxicity (post-implantation loss, resorptions) and developmental toxicity (reduced skeletal ossification and subcutaneous haemorrhage) were seen consistently; findings were apparent at dose levels of 20 mg/kg bw/d Cr (VI) and above. Information on effects of Cr (VI) on the testes is available from repeated oral dose studies. In the rat, testicular degeneration was observed at a dose level (40 mg/kg bw/d (14 mg Cr(VI)/kg bw/d) which caused a large decrease in body weight gain following gavage administration of sodium dichromate for 90 days. A NOAEL of 20 mg/kg bw/d (7 mg Cr(VI)/kg bw/d) was determined for effects on the testis.

In the study by Elsaieed and Nada (2002), effects of gestational exposure to Cr(VI) were investigated in Wistar rats. Groups of 10 pregnant rats (mean initial body weight of 170 g) were administered drinking water containing 0 or 50 mg Cr(VI)/L as potassium dichromate on days 6 to 15 of gestation. During the exposure period, dams were evaluated for clinical signs of toxicity, body weights, and food and drinking water consumption. One day before delivery, rats were sacrificed and the following were evaluated: numbers of corpora lutea, pre- and post-implantation losses, resorptions, and live and dead fetuses; fetal weight; and visceral and skeletal anomalies.

No mortalities or clinical signs of toxicity were observed. Elsaieed and Nada (2002) stated that food and drinking water consumption was comparable between control and treatment groups, although data were not reported. Gestational weight gain was significantly (40%) less in treated dams, compared with controls (p < 0.05). Based on an average gestational body weight of 177 g (average calculated using body weights at mating and at the end of gestation) and the allometric equation for drinking water consumption for laboratory mammals (0.10 × body weight0.7377; U.S. EPA, 1988), a daily dose of 7.9 mg Cr(VI)/kg bw/d was estimated. In this study, treatment of rats with Cr(VI) resulted in significant (p < 0.05) increases in post-implantation loss/litter (1.5 vs. 0), resorptions/litter (1.2 vs. 0), and dead foetuses/litter (1.2 vs. 0) and decreases in live foetuses/litter (1.5 vs. 6.8 in control) and foetal weight (33% decrease). In the exposed group, increased litters with foetal anomalies were observed including visceral (renal pelvis dilation: 2.1/litter) and skeletal (incomplete skull ossification: 1.0/litter) changes; no control foetuses showed these changes.The results of this study showed that exposure of pregnant Wistar rats to drinking water containing 50 mg Cr(VI)/L as potassium dichromate (approximately 7.9 mg Cr(VI)/kg bw/d) on days 6–15 of gestation produced adverse effects on reproductive outcome and foetal development. Thus a LOAEL of 7.9 mg Cr(VI)/kg bw/d was identified from this study.

 

Other studies found no effects on the testis, following administration of potassium dichromate by the dietary route for 9 weeks. The highest dose levels in these studies were 24 mg/kg bw/d (8 mg Cr(VI)/kg bw/d) in the rat and 92 mg/kg bw/d (32 mg Cr(VI)/kg bw/d) in the mouse (EU RAR, 2005). Summary and discussion of the available reproductive toxicity studies from the EU RAR (2005) There are three animal studies available which focus on fertility. Adverse effects were produced in mice receiving potassium dichromate for 12 weeks in drinking water at 333 mg/kg bw/d (120 mg Cr(VI)/kg bw/d) and 400 mg/kg be/d (140 mg Cr(VI)/kg bw/d) and above in males and females respectively. A NOAEL of 166 mg/kg bw/d (60 mg Cr(VI)/kg bw/d) was identified in males, but no NOAEL was found for females as 400 mg/kg bw/d was the lowest dose level tested. An increase in resorptions following treatment of males and a decrease in implantations in treated females were among the findings in this study. In another study, pregestational oral administration of potassium dichromate in drinking water to female mice produced adverse effects on fertility (reduced number of corpora lutea and increased pre-implantation loss) at 500 ppm (119 mg/kg bw/d (40 mg Cr(VI)/kg bw/d)) and above. NOAEL values of 119 mg/kg bw/d (40 mg Cr(VI)/kg bw/d) and 63 mg/kg bw/d (20 mg Cr(VI)/kg bw/d) can be identified from this study for maternal toxicity and fertility effects respectively. In a third study, also in the mouse, at 86 mg/kg bw/d (30 mg Cr(VI)/kg bw/d), the highest dose level tested, there were no effects of treatment on fertility parameters. Fetotoxicity, including post-implantation losses, has been observed in the mouse following administration of potassium dichromate in drinking water during gestation (days 0-19). Significant developmental effects occurred at the lowest dose level tested, 60 mg/kg bw/d (20 mg Cr(VI)/kg bw/d) in the absence of maternal toxicity. Therefore no developmental NOAEL was determined. Qualitatively similar results were obtained in another study in which (350 mg/kg) potassium dichromate (125 mg Cr(VI)/kg) was administered for a shorter period, on days 6-14 of gestation. In a pregestational study in female mice, fetotoxic effects were seen starting from the lowest dose level tested, 250 ppm (63 mg/kg bw/d (22.1 mg Cr(VI)/kg bw/d)) potassium dichromate. Significant levels of total chromium were found in treated animals at sacrifice. No NOAEL could be identified for the developmental effects, which included post-implantation losses. These fetal effects may possibly be explained by the presence of chromium in the dams after the end of treatment. Overall, highly water soluble chromium (VI) compounds should be considered to be developmental toxicants in the mouse.

These findings can be regarded as relevant to humans. No reproductive toxicity studies are available using the inhalation or dermal routes of exposure.

Human data relating to effects on reproduction are limited to poorly reported studies of female workers from which no conclusions can be drawn.

Justification for classification or non-classification

There is evidence that sodium dichromate effects fertility and the developing foetus in animals upon oral exposure, as discussed above. Based on this, the legal classification of sodium dichromate (Index 024 -004 -00 -7) as reprotoxic category 1B (H360FD) is adopted.