Chromium trioxide

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

A number of studies have been performed using potassium dichromate. While the studies are of non-standard design, they are considered (as a whole) to adequately assess reproductive toxicity. The results of the studies are applicable to all of the water-soluble hexavalent chromium compounds in this group.

Link to relevant study records

Reference

Reference 1

Endpoint:

fertility, other

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The effects on fertility were investigated following the administration of potassium dichromate to male and female mice for 12 weeks before mating. Treated males were mated with untreated females (experiment 1) and treated females were mated with untreated males (experiment 2). Females were sacrificed one week following mating.

GLP compliance:

not specified

Remarks:

Published study

Limit test:

no

Specific details on test material used for the study:

hexavalent chromium (potassium dichromate: Fluka AG, Chemische Fabrik CH-9470, Bucks)

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

Sexually mature male and female Swiss mice (day 50 of age) were used in these experiments. They were raised in the animal house unit in the Medical Faculty at the Jordan University of Science and Technology in controlled temperature 21°C +/- 1°C under a 12 h light: 12 h darkness schedule (lights 06.00-18.00 h). Food and water were available ad libitum.

Route of administration:

oral: drinking water

Vehicle:

water

Details on mating procedure:

1 treated male: 2 untreated females
1 untreated male: 3 treated females

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Treated males or females were mated with untreated animals following exposure for 12 weeks

Frequency of treatment:

Continuous

Details on study schedule:

The effects of potassium dichromate on male and female fertility were investigated in sexually mature (7 weeks old) 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/d (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/d (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. The Number of pregnant females, total implantations, viable fetuses 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 weights and reproductive organ weights were recorded in these animals.

Dose / conc.:

0 mg/L drinking water

Dose / conc.:

1 000 mg/L drinking water

Remarks:

males; equivalent to 166 mg/kg bw/d

Dose / conc.:

2 000 mg/L drinking water

Remarks:

males; equivalent to 333 mg/kg bw/d
females; equivalent to 400 mg/kg bw/d

Dose / conc.:

4 000 mg/L drinking water

Remarks:

males; equivalent to 666 mg/kg bw/d

Dose / conc.:

5 000 mg/L drinking water

Remarks:

males; equivalent to 833 mg/kg bw/d
females; equivalent to 1000 mg/kg bw/d

No. of animals per sex per dose:

9-20 males; 11-18 females

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

observation of mortality and clinical signs

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

not examined

Postmortem examinations (parental animals):

No of pregnant females, No of implantations, No of viable fetuses, No of mice with resorptions, Total no of resorptions were recorded. Body weights and reproductive organ weights were recorded in satellite animals

Postmortem examinations (offspring):

not examined

Statistics:

Data are expressed as means +/- S.D. Differences between control and chromium exposed groups were analyzed using either Chi-square or Student ‘t’ tests

Clinical signs:

no effects observed

Description (incidence and severity):

Generally there were no mortality or clinical signs of toxicity in any group of male or female mice exposed to hexavalent chromium compounds.

Mortality:

no mortality observed

Description (incidence):

Generally there were no mortality or clinical signs of toxicity in any group of male or female mice exposed to hexavalent chromium compounds.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight significantly decreased in 2000 mg/L and 5000 mg/L (p < 0.01) hexavalent chromium exposed males. Exposure of female mice to 5000 mg/L hexavalent chromium had no significant effect on body weights. For further details see 'Any other information on results'

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

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

When the adult males exposed to hexavalent chromium and unexposed control males were tested with adult untreated females for 5 min, males were sexually interested, as females were pursued by males, their genitalia were examined and mounting occurred.
The number of implantation sites and the number of viable fetuses 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. The numbers of resorptions and dead fetuses were increased in females impregnated by hexavalent chromium exposed males.
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). The 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. The number of viable fetuses 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.
The 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.
For further details see 'Any other information on results'

Dose descriptor:

NOAEL

Effect level:

1 000 mg/L drinking water

Based on:

test mat.

Remarks:

Potassium dichromate in drinking water (nominal); equivalent to 166 mg/kg bw/d

Sex:

male

Basis for effect level:

reproductive performance

Dose descriptor:

LOAEL

Effect level:

2 000 mg/L drinking water

Based on:

test mat.

Remarks:

Potassium dichromate in drinking water (nominal); equivalent to 400 mg/kg bw/d

Sex:

female

Basis for effect level:

reproductive performance

Critical effects observed:

not specified

Remarks on result:

not measured/tested

Reproductive effects observed:

not specified

At higher concentrations, the treated animals consumed less water per day compared to the control group (no more details provided). It is unclear whether or not the dose was adjusted for the reduced water consumption or if these animals received a lower dose. There were no deaths or clinical signs of toxicity in any group of male or female mice exposed. Compared to the control group, a statistically significant reduction in absolute body weight of 10% and 12% was seen in satellite group males at 2000 and 5000 mg/l (the only two dose levels at which body weight was recorded), respectively. Body weight of satellite group females administered 5000 mg/l potassium dichromate (the only dose at which body weight was recorded) was unaffected. Relative testes weights were statistically significantly increased at 2000 (by 17.5%) and at 5000 mg/l (by 21.5%). Relative seminal vesicles and preputial gland weights were statistically significantly reduced at 5000 mg/l only (by 27% and 34%, respectively). A statistically significant increase in relative ovarian weight (by 50%) was reported at 5000 mg/l. It is noted that in the absence of information on the absolute organ weights, the increase seen in relative testis weight could be accounted for by the reduction in absolute body weight observed in males. It is also noted that, in the absence of histopathological examinations, it is difficult to interpret the toxicological significance of these organ weight changes.

Compared to the control groups, the percentage of pregnant unexposed females mated with treated males and of pregnant exposed females mated with untreated males was unaffected by treatment. The mean number of implantation sites was statistically significantly reduced in females impregnated by males treated with 2000 (6.33 versus 8.18 in the control group) and 4000 mg/l potassium dichromate (6.86 versus 8.18), but not with the highest dose (7.84 versus 8.18). Given the absence of a dose-response relationship, the toxicological significance of this finding is uncertain. However, it is possible that at higher concentrations, the actual doses the animals received were lower than the nominal doses, due to the reduced water consumption. There were no resorptions or dead foetuses in the control groups, or in the females impregnated by males treated with 2000 or 4000 mg/l potassium dichromate. However, 3 resorptions were noted in the females impregnated by males treated with the lowest dose (1000 mg/l). Given the absence of a clear dose-response relationship and that it is not clearly reported whether these findings occurred in one single litter or in different litters, the 3 resorptions seen at 1000 mg/l are regarded as being incidental. A total number of 6 resorptions and of 6 dead fetuses was also observed in the females impregnated by males treated with the highest dose (5000 mg/l). Although it is not reported whether these findings occurred in one single litter or in different litters, given the incidence, it is unlikely they occurred in one isolated litter. Hence, the fetolethality reported at this dose level (5000 mg/l) is regarded as being treatment-related. The mean number of implantations and of viable fetuses was also statistically significantly reduced in females treated with 2000 mg/l (7.35 versus 9.00 and 6.55 versus 8.76, respectively) and 5000 mg/l potassium dichromate (7.44 versus 9.00 and 5.88 versus 8.76, respectively). There was also a statistically significant increase in the number of pregnant females with resorptions at 2,000 (53% versus 11%) and at 5000 mg/l (63% versus 11%). Similarly, a total number of 37 and 14 resorptions (versus 4 in the control group) were observed at 2000 and 5000 mg/l, respectively.

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).

Executive summary:

Male and female Swiss mice were administered potassium (VI) dichromate in drinking water for 12 weeks prior to mating with untreated animals. Female mice were sacrificed one week following treatment; the numbers of pregnant animals, total implantations, viable foetuses and resorptions were recorded. Satellite groups of animals were also treated for 12 weeks (without mating) and the weights of the reproductive organs recorded.

There were no deaths or signs of toxicity; slightly reduced bodyweights were seen in both groups of treated males. Relative testes weights were significanlty increased in both satellite groups, however findings may be secondary to bodyweight effects. Relative seminal vesicle and preputial gland weights were significantly lower, however the toxicological significance of these findings is unclear in the absence of histopathology. Relative ovary weight was increased in females at 1000 mg/kg bw/d. Mean numbers of implantation sites were lower in treated groups, however a dose-response relationship is not apparent.

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

NTP GLP guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Principles of method if other than guideline:

Task 2, the continuous breeding phase, consisted of a control group and three treated groups (20 pairs/group). F0 animals received potassium dichromate in their diet from Study Day 1 until necropsy/termination. Following seven days of premating exposure to potassium dichromate the animals were housed as breeding pairs for 85 days (12 weeks). Litters produced during the cohabitation period were counted and weighed by sex on PND 1 and then euthanized. At the end of the 85-day cohabitation period, the pairs were separated. Any litters born (F1) after the continuous breeding phase were reared by the dam until weaning on PND 21. Selected weanlings were reared in the same sex groups until PND 74±10. F1 animals received diets containing potassium dichromate
after weaning, with the same concentration of potassium dichromate as their parents received during Task 2. These F1 animals were used for assessment of second-generation reproductive toxicity (see Task 4 below). The F0 (Task 2) animals were necropsied and terminal body weights and organ weights were obtained, sperm analysis was performed and tissues were saved.
Task 4, the assessment of the F1 generation, was conducted using offspring from all four dose groups. At sexual maturity (74 ±10 days of age), twenty control animals of each sex and 20 treated animals of each sex in each dose group were randomly assigned to breeding pairs, avoiding sibling matings, and cohabitated for up to seven days then separated. Offspring were counted and weighed by sex on PND 1. At necropsy, F1 terminal body weights and organ weights were obtained, sperm analysis performed, and tissues were saved.

GLP compliance:

yes

Limit test:

no

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 study initiation: (P) study animals were approximately 10 weeks of age at the initiation of dosing; (F1) at waening (PND 21)
- Weight at study initiation: (P) Males: 21.5 - 29.9 g; Females: 15.7 - 22.2 g
- Housing: Animals were housed two per cage by sex from receipt to randomization for Task 2; two per cage (one male and one female) during Task 2 and Task 4 cohabitation; one to four per cage during the Task 4 holding and rearing phase; and housed individually at all other times. All animals were housed in polycarbonate cages suspended on stainless-steel racks with an Edstrom automatic watering system providing filtered tap water. Racks were equipped with filter paper liners. Polycarbonate caging contained Beta chip heat treated hardwood laboratory bedding.
- Diet: Powdered Zeigler™ NIH 07 Small Animal Feed was available ad libitum
- Acclimation period: 8 days
ENVIRONMENTAL CONDITIONS
- Temperature: 20-23 °C (68-74 °F)
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12-h light/12-h dark cycle was maintained throughout the study
IN-LIFE DATES: From: 05/30/95 P0 (animal arrival) To: 10/10-11/95 P0 males, 10/31/95 P0 females; 01/02-03/96 F1 males, 01/18/96 F1 females (termination)

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on mating procedure:

F0 animals were co-housed for 12 weeks (1:1). Selected F1 animals were co-housed (1:1) for up to 7 days, or until detection of a copulatory plug.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Continuous treatment. Animals were treated for one week prior to the 12-week continuous mating phase; F1 animals were treated from weaning to sexual maturity (approximately Day 74).

Frequency of treatment:

Continous (in diet)

Details on study schedule:

See above

Dose / conc.:

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

Remarks:

equivalent to 7 mg Cr(VI)/kg bw/d
F0 animals

Dose / conc.:

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

Remarks:

equivalent to 14 mg Cr(VI)/kg bw/d
F0 animals

Dose / conc.:

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

Remarks:

equivalent to 30 mg Cr(VI)/kg bw/d
F0 animals

Dose / conc.:

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

Remarks:

equivalent to 8 mg Cr(VI)/kg bw/d
F1 animals

Dose / conc.:

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

Remarks:

equivalent to 16 mg Cr(VI)/kg bw/d
F1 animals

Dose / conc.:

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

Remarks:

equivalent to 37 mg Cr(VI)/kg bw/d
F1 animals

No. of animals per sex per dose:

20

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 previous 9-week dietary study in BALB/c mice, dose levels for the continuous breeding phase for this study were set at 100, 200, and 400 ppm.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental clinical signs noted during Task 2 included abrasions. swellings, paraphimosis, ulcerations, tremors, hunched posture, languid behavior, limb paralysis, hypothermia, rapid respiration, rough haircoat, exophthalmos, opacity, vaginal discharge, thinness, tissue masses, and 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.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Mortality was observed in eight animals during Task 2 (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 treatment related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of the F0 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. Mean terminal body weights of the 400 ppm F0 females were decreased by 7% compared to the controls.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related microscopic lesions were observed in the livers or, kidneys of F0 males and females. Incidental 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 lymphocytic infiltrate of the kidneys. These lesions were not considered treatment-related due to a lack of dose response.
Because testis weight, epididymal weight and sperm count, and testicular spermatid count were all unchanged, the testis was not evaluated histologically. Since all of these measures of gametogenesis were the same as controls, there was no reason to suspect an effect on spermatogenesis.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Computer-assisted sperm analysis from vas deferens samples revealed no differences between the control and high-dose group in any of the parameters.The mean epididymal sperm density, percent abnormal sperm, and the total number of spermatids per testis were comparable among dose groups. The mean number of spermatids per mg testis was increased by 22% in the 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.
Because testis weight, epididymal weight and sperm count, and testicular spermatid count were all unchanged, the testis was not evaluated histologically. Since all of these measures of gametogenesis were the same as controls, there was no reason to suspect an effect on spermatogenesis.

Description (incidence and severity):

During the lactation phase of the final litter, the mean average pup weights, proportion of pups born alive, and pup survival of the F1 animals were generally comparable among groups

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

incidental clinical signs noted during Task 4 included paraphimosis, hunched posture, languid bebavior, hypothermia, and thinness. These observations were mainly for one control male which was killed in extremis on PND 84. The incidence of these observations was low (5% per group) and no dose-related differences were observed.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Mortality was observed in twenty-nine animals during the Task 4 bolding 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 (PND 22-29). The cause of death for these 18 animals was most likely due to weanlings being separated from the mothers at PND21. The weanlings weighed less than 10 grams at PND 21 and between 17-23 grams at PND 74±10. If the pups had remained with the dam until PND 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 Task 4 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

Description (incidence and severity):

No differences were observed in the mean dam or sire weights obtained following delivery. Terminal body weights of the F1 males and females were comparable among the dose groups.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No differences were observed in any of the absolute organ weights. Only one incidental change was noted in the relative organ weights (mg/g body weight): the mean kidney-to-body weight ratio was increased by 5% in the 200 ppm females.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Only one control male displayed liver foci.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related microscopic lesions were observed in the livers or kidneys of F1 males and females. Incidental microscopic lesions included hepatocyte cytoplasmic vacuolization; individual cell necrosis of hepatocytes; necrosis of the liver; and renal tubule degeneration, regeneration, and dilatation. These lesions were not considered treatment-related due to a lack of a relationship to dose.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Evaluation of the vaginal smears revealed no differences between the groups in the amount of time spent in the different estrous stages, cycle length, number of cycles, number of cycling females, or number of females with regular cycles

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Evaluation of the computer-assisted sperm motion parameters revealed no differences between the control and high-dose group. Because of this lack of difference, the middle and low-dose group samples were not evaluated. 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:

no effects observed

Description (incidence and severity):

Measures of reproductive performance of second generation breeding pairs revealed no differences between dose groups in mating index, pregnancy index, fertility inidex, number of live pups per litter, proportion of pups born alive, sex ratio of pups, adjusted live pup weight, and gestation length. 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.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

not specified

There was no effect due to treatment on fertility in the F0 animals. The number of dams delivering/number cohabiting was comparable between the groups. There were no differences due to treatment in the mean number of litters/pair, number of live pups/litter, sex ratio, and absolute or adjusted live pup weight. Gestation length was not affected. No signs of toxicity were observed. However, a slight decrease (10% at week 14) in body weights occurred in F0 females at 85.7 mg/kg bw/d (30 mg Cr(VI)/kg bw/d). At necropsy, F0 liver weights were decreased by 17% (males) and 12% (females) at the highest dose level. No treatment-related gross or microscopic lesions occurred in F0 animals. The results of the sperm analyses were comparable between groups. At the highest dose level, mean pup weight was slightly less than control values (9-15%, not statistically significant) at days 14 and 21 after birth. By day 74, the difference remained at 9%. Mean average pup weights during lactation, proportion of pups born alive and survival of F1 pups was generally comparable throughout the groups. No treatment-related signs of toxicity were observed in F1 adults. There were no treatment-related effects on reproductive performance (mating index, pregnancy index, fertility index, number of live pups/litter, proportion of pups born alive, sex ratio, adjusted live pup weight and gestation length) in the F1 breeding phase. Vaginal smear results indicated that there were no effects on oestrus cyclicity. No treatment-related changes in organ weight or sperm analysis data and no treatment-related gross or microscopic lesions occurred in the F1 groups at necropsy. Live female pup weight was decreased by 11% at the highest dose level. There were no differences in F2 number of live and dead pups per litter, sex ratios or pup weights between exposed groups and controls.

Conclusions:

No evidence of any reproductive toxicity was seen under the conditions of this study, at dose levels of up to 30 mg/kg bw/d Cr (VI).

Executive summary:

The reproductive toxicity of potassium dichromate was investigated in a two-generation study in the 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. 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.

Effects of toxicity were limited to reduced weight gain and liver weights at the top dose level equivalent to 30 mg/kg bw/d Cr (VI). No evidence of an effect on fertility was seen in either generation.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

40 mg/kg bw/day

Study duration:

subchronic

Species:

mouse

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 an 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).

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. 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 bw/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. It is noted that some of the adverse effects on reproduction observed in animal studies may be related to the germ cell mutagenicity of these chromium (VI) compounds (see Mutagenicity section). 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

Description of key information

A number of studies have been performed using potassium dichromate. While the studies are of non-standard design, they are considered (as a whole) to adequately assess developmental toxicity. The results of the studies are applicable to all of the water-soluble hexavalent chromium compounds in this group.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-standard published study; reported in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

In a study specifically performed to assess the effect of pre-gestational exposure to chromium on development, groups of 15 female Swiss albino mice of proven fertility were administered daily 0, 250, 500 or 750 ppm potassium dichromate (equivalent to doses of approximately 0, 63, 119 and 174 mg/kg/day (0, 20, 40 and 60 mg Cr(VI)/kg/day) in drinking water for 20 days. The animals were then immediately mated for 24 hours with untreated males, and, subsequently, 10 pregnant females were randomly selected from each group and sacrificed on day 19 of gestation. Both ovaries were removed from the dams to determine the number of corpora lutea. Numbers of implantations and resorptions were recorded and the fetuses were subjected to routine external, visceral and skeletal examination. In addition, at sacrifice, levels of total chromium in the maternal blood, in the placenta and in the fetal tissues were measured.

GLP compliance:

no

Remarks:

Published study

Limit test:

no

Species:

mouse

Strain:

Swiss

Details on test animals or test system and environmental conditions:

Sixty, 4-month old, Swiss albino, female mice (body weight 30 +/- 5 g) of proven fertility from the Industrial Toxicology Research Centre colony were divided into four groups of fifteen mice each. The animals were individually housed under standard animal house conditions (room temperture 20-22°C, relative humidity 50+/-5%) where a regular cycle of 12 hrs light: 12 hrs darkness was maintained and were provided with feed pellets (Lipton India Ltd.) and water ad libitum.

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

Females were administered potassium chromate in the drinking water for 20 days, prior to mating

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Exposed females were mated for 24 hours with untreated males

Duration of treatment / exposure:

Females were administered potassium chromate in the drinking water for 20 days, prior to mating

Frequency of treatment:

Continous (in drinking water)

Duration of test:

Females were treated for 20 days prior to a 24-hour mating period with untreated males. Pregnant females were sacrificed on Day 19 of gestation.

Dose / conc.:

250 ppm

Remarks:

equivalent to approx. 63 mg/kg/day and 20 mg Cr(VI)/kg/day

Dose / conc.:

500 ppm

Remarks:

equivalent to approx. 119 mg/kg/day and 40 mg Cr(VI)/kg/day

Dose / conc.:

750 ppm

Remarks:

equivalent to approx. 174 mg/kg/day and 60 mg Cr(VI)/kg/day

No. of animals per sex per dose:

15 females were treated and mated; 10 pregnant animals were randomly selected for further investigation

Control animals:

yes, concurrent vehicle

Details on study design:

The selection of doses was based on our earlier study (Trivedi et al. 1989) and the fact that the average chromium intake of humans is approximately 200 g/day in drinking water (NRC 1989)

Maternal examinations:

Maternal investigations were limited; blood levels of chromium were investigated

Ovaries and uterine content:

Both ovaries were removed from the dams to determine the number of corpora lutea; the numbers of implantations were recorded

Blood sampling:

Known amounts of maternal blood (withdrawn from the heart of 5 animals/group), of placentae and the fetuses were digested in Nitric acid:Perchloric acid (6:1) mixture till a white residue remained at the bottom of the flask. The residue was dissolved in 5.0 ml of 0.1 N Nitric acid and read on DC Plasma Emission Spectrophotometer (Beckman Spectrospan V). Blank and spiked samples were also run and analyzed simultaneously

Fetal examinations:

Foetuses were assessed for external, visceral and skeletal findings.

Statistics:

Embryo- and feto-toxicity data and chromium estimation data were analysed by one-way ANOVA followed by Student's 't' test while gross and skeletal abnormalities data were analysed by Fischer's Exact Test

Clinical signs:

no effects observed

Description (incidence and severity):

No signs of toxicity were observed in any of the treated females.

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality of 20% (3 of 15 females) was observed in the top dose group. Although autopsy of these animals could not establish the cause of death, given the number of deaths and the fact that they occurred at the highest dose level, they are likely to be treatment-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain was unaffected during the treatment period, however during gestation, almost no body weight gain was seen in the top-dose dams, and a reduction in body weight gain of 14% was observed in the mid-dose dams.

Description (incidence and severity):

Daily chromium (VI) intake as calculated by water consumed: 1.9 ± 0.02, 3.56 + 0.03, and 5.23 + 0.07 mg Cr for groups II, III, and IV, respectively. Water consumption in the control group was 8.52 + 0.21 mL/mouse/day,

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Compared to controls, a statistically significant reduction in the number of corpora lutea of 44% was noted at 750 ppm. Also, no implantations were seen in this group. The number of implantations was also statistically significantly reduced (by 29% of the control value) in the dams pre-gestationally treated with 500 ppm potassium dichromate. A dose-related (statistically significant in the mid-dose group) increase in pre-implantation loss was seen at 250 and 500 ppm. Statistically significantly increased incidences of post-implantation losses were observed at 250 and 500 ppm, and of resorptions at 500 ppm.

Dose descriptor:

LOAEL

Effect level:

20 mg/kg bw/day

Based on:

element

Remarks:

equivalent to 250 ppm test material

Basis for effect level:

other: developmental toxicity

Dose descriptor:

LOAEL

Effect level:

750 ppm

Based on:

test mat.

Remarks:

equivalent to 174 mg/kg bw/day (60 mg CrVI/kg bw/day)

Basis for effect level:

body weight and weight gain

Dose descriptor:

LOAEL

Effect level:

500 ppm

Based on:

test mat.

Remarks:

equivalent to 119 mg/kg bw/day (40 mg CrVI/kg bw/day)

Basis for effect level:

pre and post implantation loss

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Fetal weight and crown-rump length were statistically significantly reduced in the low- and mid-dose groups.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

There was also a dose-related (statistically significant in the mid-dose group) reduction in litter size at 250 and 500 ppm.

External malformations:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increased incidence of kinky tail, short tail and subdermal hemorrhagic patches was seen at 500 ppm.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant reduced ossification in the parietal, interparietal and caudal bones was observed in fetuses of dams pregestationally treated with 500 ppm. Fetal caudal ossification was also significantly reduced at 250 ppm.

Visceral malformations:

no effects observed

Description (incidence and severity):

No significant abnormalities were seen during soft tissue examinations in any of the treated groups.

Description (incidence and severity):

Total chromium levels were significantly increased above levels in the control group for the maternal blood in all the treated groups, for the placenta at 250 and 500 ppm and for the fetal tissues at 500 ppm.

Dose descriptor:

LOAEL

Effect level:

250 ppm

Based on:

test mat.

Remarks:

equivalent to 63 mg/kg bw/day (20 mg CrVI/kg bw/day)

Sex:

male/female

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Chromium-induced embryo- and feto-toxicity in mice treated during pregestational period

Parameters	Group I (control)	Group II (250 ppm)	Group III (500 ppm)	Group IV (750 ppm)
Weight gain in mothers (g)	14.40 ± 1.01	13.43 ± 0.50	12.38 ± 0.49	1.7 ± 0.93
Number of corpora Iutea/mice	7.9 ± 1.01	7.4 ± 0.50	7.3 ± 0.37	4.4 ± 0.50abc*
Number of implantations/mice	7.7 ± 0.74	6.8 ± 0.41	5.4 ± 0.27a*	0
Number of live fetuses/mice	7.7 ± 0.74	5.8 ± 0.50	3.4 ± 0.24ab*	0
Number of resorptions/mice	0	1.20 ± 0.44	2.0 ± 0.31a*	0
Pre-implantation loss (%)	2.77 ± 1.21	8.38 ± 3.53	24.79 ± 2.17ab*	100
Post-implantation loss (%)	0	17.51 ± 2.22a*	36.66 ± 4.94ab*	0
Fetal weight (g)	1.59 ± 0.04	1.11 ± 0.04a*	0.97 ± 0.03ab*	0
Placental weight (g)	0.137 ± 0.003	0.128 ± 0.005a*	0.223 ± 0.005ab*	0
Crown-rump length (cm)	2.92 ± 0.07	2.41 ± 0.08a*	2.09 ± 0.08ab*	0

Value represents mean± S.E. of 10 female mice in each group.

The significance of the difference among various groups was evaluated by applying one-way ANOVA followed by Student’s ‘t’ test. * Significance p< 0.05. Comparison between two groups: a –vs. control; b –vs. 250 ppm; c –vs. 500 ppm

Incidence of gross and skeletal abnormalities in the pups of dams treated with chromium during the pregastational period

Parameters	Group I (control)	Group II (250 ppm)	Group III (500 ppm)
Gross abnormalities
Number of pups/litter observed	72/10	51/10	19/10
Drooping wrist	0/10	0/10	6/4 (32)
Sub-dermal hemorrhagic patches	0	8/6 (16)	8/4 (42)a*
Kinky tail	0	0	8/6 (42)a*
Short tail	0	4/4 (9)	10/4 (53) a*
Skeletal abnormalities
Number of pups/litter observed	43/10	34/10	19/10
Reduced parietal ossification	0	0	12/10 (63)a*
Reduced interparietal ossification	0	0	10/10 (53)a*
Reduced caudal ossification	6/4 (12)	18/8 (53)a*	18/10 (95)a*

Gross and skeletal abnormalities are represented as number of abnormal pups/litters observed. The statistical significance was evaluated by Fisher’s Exact test. Percentage on parentheses calculated by the total number of pups observed. * Significance p< 0.05. Comparison between two groups: a –vs. control.

Conclusions:

Exposure of female mice to potassium chromate prior to mating resulted in reduced fertility (decreased numbers of corpora lutea, increased implantation loss) and also in developmental toxicity. Maternal toxicity was seen at the top dose level (mortality).

Executive summary:

Female mice were exposed to potassium dichromate in drinking water at levels of 0, 250, 500 or 750 ppm (equivalent to Cr (VI) intakes of 0, 20, 40 and 60 mg/kg bw/d) for 20 days prior to mating with untreated males. Dams were sacrificed at Day 19 of gestation and the foetuses examined.

Deaths occurred at the top dose level of 60 mg/kg bw/d; no additional maternal toxicity was apparent. At 60 mg/kg bw/d there were reduced corpora lutea and no implantations; implantation numbers were also reduced at 40 mg/kg bw/d. Pre-implantation loss was increased at 20 and 40 mg/kg bw/d; increased post-implantation losses were also seen at 20 and 40 mg/kg bw/d and resorptions were increased at 40 mg/kg bw/d.

Foetal numbers were significantly lower and foetal weight and size were significantly lower at 20 and 40 mg/kg bw/d.

There was no evidence of teratogenicity. Increased incidences of kinky tail, short tail and subcutaneous haemorrhage were seen at 40 mg/kg bw/d. Reduced ossification of a number of bones was seen at 40 mg/kg bw/d; findings at 20 mg/kg bw/d were limited to reduced caudal ossification. Analysis of chromium levels revealed distribution to the maternal blood, placenta and foetal tissue at 40 mg/kg bw/d.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

20 mg/kg bw/day

Study duration:

subacute

Species:

mouse

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

One developmental toxicity study on chromium trioxide is available investigating embryotoxic effects in hamsters. No other studies of the developmental toxicity of chromium (VI) trioxide have been identified, however a number of studies performed with potassium dichromate are available. Once systemically absorbed, the toxicokinetics and toxicodynamics of chromium (VI) trioxide and the other water-soluble Cr (VI) salts are essentially identical, therefore the results of these studies are relevant and can be extrapolated. The available studies investigated both reproductive developmental toxicity endpoints and are discussed above.

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.

Justification for classification or non-classification

Chromium (VI) trioxide, sodium chromate, sodium dichromate and potassium dichromate are classified under Annex I to directive 67/548/EEC. Chromium (VI) trioxide is listed in Annex VI to Regulation (EC) No 1272/2008 under Index No 024-001-00-0 with the following harmonised classification:

Repr. 2, H361f ‘Suspected of damaging fertility’

No change to this classification is proposed.