Reaction mass of sodium chloride and potassium chloride and trisodium hexafluoroaluminate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

There is no indication for fertility risks caused by cryolite.

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, unpublished report available, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Kryocide (Cryolite); Sodium aluminofluoride
Supplier: Elf Atochem North America, Inc.
Batch number: 86.12
Purity: 96.0%

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals: albino rat
Strain: CD (Sprague Dawldey derived)
Age at receipt: 30 days
Supplier: CharlesR iver Laboratories, Inc. Portage, Michigan 49081
Animals were acclimated for approximately two weeks.
Housing: Suspended, stainless steel, wire mesh bottom cages. Animals were housed individually throughout the study with the following exceptions: the first week of acclimation( two animals/sex/cage); mating (one male and one female/cage overnight); lactation( dam and litter/cage) and post-weaninogf the FI pups (one- two llttermates/sex/cage until formali nitiation of the pre-matingt reatmentp eriod which followed weaning of the last litters).
Feed and water: Ad libitum. Certified Rodent Diet No. 5002 (mash) was provided fresh at least weekly during the study. Analysis of each feed lot used during this study was performed by the supplier (Purina Mills, Inc. St. Lous, MO).

Environmental conditions: 12 hour light/dark cycle, actual temperature: 64-80°F (18-27°C), actual relative humidity: 24-79%.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

Appropriate amounts of the test substance were mixed with the basal granulated diet to achieve the desired concentrations (prepared at a fixed concentration [ppm] in the diet and no correction for purity of the test substance was used). Fresh diets were prepared and presented to animals weekly during the study. Control animals received standard laboratory diet only. Unused portions of diet (control and treated) were stored at ambient temperature during intervals when not being presented to animals.

Prior to the initiation of the study, full-size (mock) batches of diets at the low- and high concentration levels( 200 and 1800 ppm, respectively) were evaluated to determine that diets prepared using the proposed mixing procedure were homogeneous. To determine homogeneity, three randomly drawn diet samples (approximately 100 grams each) were taken from each mix at each of three levels (top, middle, bottom - total of nine samples) in the mixer. If the data demonstrated that the mean of the values for the three levels were within ±15% of each other and ±20% of the nominal (desired) concentration, the batches were considered homogeneous.

In previous studies conducted in this Testing Facility with dietary administration of Kryocide, the stability of the Kryocide in the diet for 14 days post-preparation at ambient storage was established at dose levels that ranged from 50 to 5000 ppm. On the basis of these stability data, the only additional stability assessment performed for this study was a 21-day stability at ambient storage. These analyses were performed at the low- and high- concentrations (200 and 1800 ppm, respectively) for diets prepared to establish homogeneity. Following 21 days of storage at ambient temperature, two samples of approximately 100 grams each were collected at each concentration level and analyzed

Details on mating procedure:

Initially, one male was co-housed with one female from the same treatment group nightly until evidence of mating was observed or for seven consecutive days. Each morning following cohabitation with the male, the female was evaluated for evidence of mating (microscopic observation of sperm in the vaginal smear and/or copulation plug in the vagina). The day on which evidence of mating was observed was defined as Day 0 of gestation. Once mated, females were removed from the mating unit and housed individually for the remainder of gestation. Females unmated after the initial seven-day mating interval were redistributed (randomly) to a different male within the same treatment group until evidence of mating was observed or for seven additional days. This same procedure was repeated for a third seven-day mating interval for unmated females. This same mating design was used for F1 generation. In the mating assignments of the F1 generation, care was taken in establishing the mating units to avoid brother-sister matings.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Two diet samples of approximately 100 grams each were collected for each test diet and control at the weekly preparation intervals through out the study. Analyses to confirm concentration levels of diets intended for use on study were performed for the first four mixes (Study Weeks 1-4) and subsequently for every fourth mix (Study Weeks 8, 12, 16, 20. 24. 28, 32, 36, 40, 44 and 48) for the remainder of the study. Samples collected from mixes not scheduled for analyses have been retained frozen at this testing facility until issuance of the final report.
Diet samples analyzed were evaluated in duplicate. The diets were considered acceptable for study if values for the duplicate assays were within ± 20% of each other and the mean of the two values were within ± 20% of the nominal (desired) concentration. All diet analyses (i.e., homogeneity, stability and confirmation of concentration levels) were performed by the Analytical Department of Pharmaco LSR, Toxicology Services North America.

Duration of treatment / exposure:

F0 generation animals received the appropriate treated diets for 14 weeks prior to initiation of mating (i.e., pre-mating period) and treatment continued until sacrifice. Following the formal initiation of the pre-mating period, F1 generation animals received the appropriate treated diets for 14 weeks prior to initiation of mating and treatment continued until sacrifice. F1 pups consumed diets at the treatment level of the dam late in lactation and selected F1 animals continued to consume diets at these concentration levels during the post-weaning period through to the formal initiation of the pre-mating period.

Frequency of treatment:

continuous

Details on study schedule:

Both parental generations received a 14-week pre-mating period and treatment continued during a 21-day mating period and post-mating interval (males and unmated females) until sacrificed. Mated females continued to be treated during the ensuing gestation, lactation and post-weaning periods until sacrifice.

Remarks:

Doses / Concentrations:
14, 42, and 128 mg/kg bw/day for males and 16, 49, and 149 mg/kg bw/day for females, respectively, during premating
Basis:

Remarks:

Doses / Concentrations:
200, 600 and 1800 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

30

Control animals:

yes, plain diet

Details on study design:

Parental animals (F0, F1) were observed twice daily for mortality and unusual findings and each animal received a detailed physical examination weekly. Body weights and food consumption for the parental animals were recorded weekly during the pre-mating treatment periods and these parameters continued to be recorded weekly for males during the mating and post-mating period through to sacrifice. Body weights and food consumption were recorded for mated females at regular intervals during the gestation period and females with litters continued to be weighed at regular intervals during the lactation period. Each parental generation produced a single litter and pups were weaned on lactation day 21. On lactation day 4, litters with greater than eight pups were culled to that number so as to equalizes sex distribution (four/sex), when possible; litters with less than eight pups at Day 4 were not adjusted. During lactation, litter size, pup weights and pup sex distribution data were recorded. Randomly selected pups from the F1 litters (at least one pup/sex/litter) were chosen to become the F1 parental generation. At sacrifice, parental animals were given a gross postmortem examination and reproductive tissues, pituitary glands and gross lesions were taken and preserved in 10% neutral buffered formalin. During the sacrifice of the F1 parental males, testes and epididymal weights were also recorded. Reproductive tissues and pituitary glands were evaluated histomorphologically for control and high-dose animals from both parental generations and gross lesions with the exclusion of incisor observations, were evaluated for all animals.

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes (observations of mortality and gross clinical findings)
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes (Detailed physical examination for signs of local or systemic toxicity, pharmacological effects and palpation for tissue masses).
- Time schedule: weekly

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes. Compound intake was calculated using food consumption and the nominal concentration in the diet

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes]
- Maximum of 8 pups/litter
PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
-number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities
GROSS EXAMINATION OF DEAD PUPS:
-yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

Postmortem examinations (parental animals):

At sacrifice, parental animals w ere given a gross postmortem examination and reproductive tissues, pituitary glands and gross lesions were taken and preserved in 10% neutral buffered formalin. During the sacrifice of the F1 parental males, testes and epididymal w eights w ere also recorded. Reproductive tissues and pituitary glands were evaluated histomorphologicallfy or control and high-dose animals from both parental generations and gross lesions w ith the exclusion of incisor observations, were evaluated for all animals.

Postmortem examinations (offspring):

The unselected F1 pups and all F2 pups were sacrificed at weaning or shortly thereafter, and evaluated for external and internal irregularities and only abnormal tissues were saved (10% neutral buffered formalin).

Statistics:

Several statistical methods were used, dependent on the parameter studied.

Reproductive indices:

Mating, pregnancy and fertility rates; mean number of days-to-mating

Offspring viability indices:

percentage of pups alive at day 4

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

200 and 600 ppm:
No adverse effects of treatment at dietary levels of 200 or 600 ppm were evident from parental or neonatal parameters.
During the detailed physical evaluations of the parental animals, white discoloration of the incisors (upper and/or lower) was seen in both generations at both concentration levels and at the 600 ppm level, additional observations involving the incisors i.e., presence of a beveled edge along the anterior margin and/or a mottled appearance, were seen with increased frequency. These effects which were considered indicative of a treatment-related response were almost identical to toxic effects noted with excessive fluoride exposure i.e., dental fluorosis. This was not entirely unexpected considering the chemistry of the test material. No changes in the critical reproductive indices were seen at the low- and mid-dose levels during either generation and the effects seen were clearly non-reproductive organ effects involving the teeth.

1800 ppm:
At the 1800 ppm dietary level, no adverse effects of treatment were evident in the parental generation animals (F0, F1). The only treatment-related responses seen in these parental-animals involved observations to the incisors similar to those discussed above. All of these types of observations were seen with increased frequency at this treatment level. Bevelled anterior edge of the lower incisor was observed in 67% of animals from both generations at 1800 ppm.
No adverse effect of treatment at the 1800 ppm dietary level was evident from the macroscopic evaluations of the parental animals. Likewise, microscopic evaluation of the reproductive tissues and pituitary glands of the parental animals (F0, F1) revealed no adverse effects. Additionally, microscopic evaluation of the incisors of several F1 males with findings at necropsy i.e., white discoloration, beveled edges, and other gross lesions for both parental generations revealed no adverse effect of treatment.

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

600 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: decreased mean body weights during lactation for both the F1 and F2 litters and an increased incidence of F1 and F2 animals with pale/white livers and/or kidneys and enlarged hearts at weaning

Remarks on result:

other: Generation: F1 and F2 (migrated information)

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

1 800 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: no effects on fertility were observed

Remarks on result:

other: Generation: P and F1 (migrated information)

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

200 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: dental fluorosis

Critical effects observed:

no

Lowest effective dose / conc.:

200 ppm

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

no effects observed

At the 1800 ppm dietary level, a decrease in mean body weights during lactation for both the F1 and F2 litters (significantly decreased pup body weights during lactation days 7, 14, and 21 (82%-88% of control in F1 offspring) and days 4, 7, 14, and 21 (74%-89%) of control in F2 offspring) and an increased incidence of F1 and F2 animals with pale/white livers and/or kidneys and enlarged hearts at weaning.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 800 ppm

Sex:

male/female

Basis for effect level:

other: no effects on fertility were observed

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

1 800 ppm

Sex:

male/female

Basis for effect level:

other: no effects observed

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

128 mg/kg bw/day

Study duration:

subchronic

Experimental exposure time per week (hours/week):

168

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Additional information

In a two-generation reproduction study, Sprague-Dawley rats (30 per group) were administered cryolite in the diet at dose levels of 0, 200, 600, or 1800 ppm (representing 0, 14, 42, and 128 mg/kg/day for males and 0, 16, 49, and 149 mg/kg/day for females, respectively, during premating) (Pharmaco LSR, Inc., 1994). Compound-related systemic toxicity was observed in a dose related manner among both sexes and generations at all dose levels as evidenced by clinical signs of dental fluorosis. Whitening of the upper and/or lower incisors was observed in most treated animals of both generations. Bevelled anterior edge of the lower incisor was observed in 67% of animals from both generations at 1800 ppm. Mottled appearance of the lower incisor was noted at dose levels ≥ 600 ppm in 6%-40% of F1 animals; however, this sign was not dose related.

Reproductive toxicity was observed at 1800 ppm as evidenced by significantly decreased pup body weights during lactation days 7, 14, and 21 (82%-88% of control in F1 offspring) and days 4, 7, 14, and 21 (74%-89%) of control in F2 offspring). Gross findings were also observed in pups from both generations at 1800 ppm by the time of weaning. They were manifested as pale kidneys, pale livers and enlarged hearts and were considered to be compound related. No effects were observed on parental reproductive performance.

From this study a NOAEL for fertility of >1800 ppm / >128 mg cryolite/kg bw/day can be derived.

Besides dental fluorosis and teeth whitening, no other compound-related systemically toxic effects could be revealed from this study with daily dosages of up to and including 128 mg cryolite/kg bw/day.

Based on the observed decreased mean body weights during lactation for both the F1 and F2 litters and an increased incidence of F1 and F2 animals with pale/white livers and/or kidneys and enlarged hearts at weaning at 1800 ppm, a NOAEL for developmental toxicity of 600 ppm / 42 mg cryolite/kg bw/day is derived from this study (see also section on developmental toxicity).

As there is no indication for fertility risks caused by cryolite, a quantitative assessment for effects on fertility is not necessary.

Effects on developmental toxicity

Description of key information

During the two-generation study with rats, effects on postnatal growth evidenced by significantly decreased pup body weights during lactation as well as pathologic gross findings in several organs of the pups resulted from dose levels without any significant parental toxicity. Because these effects occurred without any significant sign for parental toxicity it is considered indicative for a specific toxic potential of cryolite adverse to postnatal development. The NOAEL for these effects in this study was 42 mg cryolite/kg bw/day

Additional information

Cryolite was investigated for prenatal developmental toxicity in rats, mice and rabbits with the oral route of administration. While in the studies with rats and with rabbits no evidence of prenatal developmental toxicity was observed, some indications of bent ribs and bent limb bones were reported in the mice study.

 

Cryolite was administered by gavage in the developmental toxicity study to female CD-1 mice (25/group) at dose levels of 0, 30, 100 or 300 mg/kg bw/day once daily from gestation days 6 through 15 (WIL Research Laboratories, 1991). There was increased mortality at 300 mg/kg bw/day. The glandular portion of the stomach was red beginning at 100 mg/kg bw/day. In addition, females in the 300 mg/kg bw/day group exhibited dark red contents of the stomach.

Fetuses at 300 mg/kg bw/day exhibited bent ribs and bent limb bones.

The study reveals severe maternal toxicity in terms of mortality and signs of toxicity in the gastrointestinal tract induced at dosages of 100 mg cryolite/kg bw/day leading to derivation of a NOAEL for maternal toxicity of 30 mg cryolite/kg bw/day.

Based on the observation of skeletal anomalies at the dose level of 300 mg cryolite/kg bw/day also a NOAEL for developmental toxicity of 100 mg cryolite/kg bw/day can be derived from the study. As these anomalies were only reported at dose levels showing severe maternal toxicity, the effects are not considered to be indicative for a substance specific teratogenic potential of cryolite.

 

During the two-generation study with rats (Pharmaco LSR, Inc., 1994), effects on postnatal growth evidenced by significantly decreased pup body weights during lactation as well as pathologic gross findings in several organs (kidney, liver, heart) of the pups resulted from dose levels without any significant parental toxicity. Because these effects occurred without any significant sign for parental toxicity it is considered to be indicative for a specific toxic potential of cryolite adverse to postnatal development. The respective NOAEL for these effects in this study was 42 mg cryolite/kg bw/day.

Based on the available data, the endpoint developmental toxicity is not critical compared to repeated dose toxicity for cryolite, i.e., the DNEL derived for repeated dose toxicity is considered the most critical one in quantitative terms:

DNEL inhalation for workers based on developmental toxicity NOAEL of 42 mg/kg bw/day:

42 mg/kg bw/day / 0.38 m3/kg bw * 85/100 * 6.7 m3/10 m3 / (2.5*5) = 5 mg/m3.

The inhalation DNEL for workers based on the local effects after repeated exposure is 0.1 mg/m3 (see DNEL section).

Justification for classification or non-classification

In accordance to Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification is not necessary for effects on fertility.

Effects adverse to development were identified from the two-generation study with rats. The critical adverse effects that had been revealed were impairment of postnatal growth evidenced by significantly decreased pup body weights during lactation as well as gross pathological changes in several organs (kidney, liver, heart) of the pups (not statistically significantly increased) observed at dose levels of 128 mg/kg bw/day without any significant maternal toxicity. No effects were observed that could indicate that pups under lactation were already affected in utero (normal weight at birth and absence of visual defects). From the data that are available it could be concluded that the observed effects in the pups were due to the lactation itself. The effects may have been caused by reduced amount of milk available to the pups or the altered nutritional composition of the milk or by the presence of fluoride in the milk. Therefore, cryolite is proposed to be classified with R64 in accordance to Directive 67/548/EEC and H362 (May cause harm to breast-fed children) under EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008).