(1-hydroxyethylidene)bisphosphonic acid, potassium salt

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See read-across justification in IUCLID Section 13

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

447 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed

Remarks on result:

other: equivalent to 368 mg/kg bw/day active acid

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

447 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed

Remarks on result:

other: equivalent to 368 mg/kg bw/day active acid

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

447 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Remarks on result:

other: equivalent to 368 mg/kg bw/day active acid

Key result

Reproductive effects observed:

no

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1971

Report date:

1970

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

Use of 2 dose levels; Sexual milestones in pups/analytical confirmation of dose not given; No sperm analyses (but conception rate of 90% suggests unaffected spermatogenesis, and OECD 416 allows omission if information is available from e.g. 90 day study)

GLP compliance:

no

Remarks:

prior to GLP

Limit test:

no

Test material

Test material information

1

Reference substance name:

Sodium Salts of (1-Hydroxyethylidene)bisphosphonic acid (2-3 Na:1)

EC Number:

701-238-4

Molecular formula:

HEDP-2Na C2H6Na2O7P2 HEDP-3Na C2H5Na3O7P2

IUPAC Name:

Sodium Salts of (1-Hydroxyethylidene)bisphosphonic acid (2-3 Na:1)

Test material form:

not specified

Test animals

Species:

rat

Strain:

other: Charles-River

Sex:

male/female

Details on test animals or test system and environmental conditions:

Weanling Charles-River rats were distributed into 5 groups, each composed of 22 females and 22 males, according to body weight and litter. They were housed in stainless steel cages with food (Ground Purina Laboratory Chow) and fresh water furnished ad libitum. Room temperature was maintained at 23 +/- 1°C, and relative humidity at 50 +/- 5%. Lighting was on a 12 hour cycle and background music was employed to equalize ambient noises.
During the first 8 weeks, the rats were caged individually and feed consumption and body weights were recorded at weekly intervals. The rats were then paired and placed into mating cages. This procedure was repeated for the breeders in the second generation (F1b).
Pregnant females were placed in nesting cages which were furnished with a plywood liner and shredded paper.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

When appropriate, HEDP-2Na was mixed with the diet at levels of 0.5 and 0.1%. Both sexes were fed with the respective diets either continuously or only during the time when the females were in their sixth through fifteenth days of gestation.
Females have been determined to be in pro-estrus by vaginal smearing and continuous exposure started "from weaning", i.e. in week 5.

Details on mating procedure:

After 8 weeks, the rats were paired and placed into mating cages. This procedure was repeated for the breeders in the second generation (F1b).

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Exposure period: continuous

Frequency of treatment:

daily

Details on study schedule:

When appropriate, HEDP-2Na was mixed with the diet at levels of 0.5 and 0.1%. Both sexes were fed with the respective diets either continuously or only during the time when the females were in their sixth through fifteenth days of gestation.
During the first 8 weeks, the rats were caged individually and feed consumption and body weights were recorded at weekly intervals. The rats were then paired and placed into mating cages. This procedure was repeated for the breeders in the second generation (F1b).

Pregnancies were timed by vaginal smearing (Long and Evans, 1922), and identification of sperm in the smear designated day 0. The pregnant females were then placed in nesting cages which were furnished with a plywood liner and shredded paper.

The original females (F0) were allowed to deliver their first 2 litters, while the third litters were used for teratologic evaluation. The newborns were counted at birth, but not handled until they were 4 days old. They were then weighed, sexed and inspected grossly. All litters containing more than 8 pups were reduced to that number to equalize the stress on the mothers during the lactation period. After the weaning, weights were recorded, all F1a pups were discarded, but 25 weanlings of each sex, from each treatment group, were selected from the F1b litters for second generation breeding stock.

Twenty (20) pairs of rats from each group were bred and the remaining 5 pairs were necropsied and examined histologically. Subsequently, the first litters of the second generation (F2a) were evaluated similarly to the F1a pups, while the second litters (F2b) were used for teratologic evaluation.
During the teratological phases one-half of each treatment group, selected prior to mating, were sacrificed by excessive ether-inhalation on day 13 and the other half on day 21 of the gestation period.

The pregnant females were examined for number of resorptions, corpora lutea and implantations. The foetuses were dried of amniotic fluid, sexed, carefully inspected for gross abnormalities and weighed. One-third of the foetuses were cleared, stained with Alizarin red stain (Staples and Schnell, 1964) and examined for skeletal defects. The remaining two-thirds of the foetuses were examined for soft-tissue anomalies, either by histological methods or by freehand sectioning (Wilson, 1965).

In addition to the post weaning F1b rats, selected organs from 5 dams from each treatment group were evaluated histologically during each teratological phase.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.1 other: %

Remarks:

nominal in diet; equivalent to 112 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

0.5 other: %

Remarks:

nominal in diet; equivalent to 447 mg/kg bw/day

No. of animals per sex per dose:

22 (F0)
25 (F1b) - 20 for breeding F2 generation and 5 for histological examination

Control animals:

yes, plain diet

Examinations

Parental animals: Observations and examinations:

Feed consumption and body weights were recorded at weekly intervals.
Evaluation of conception rate, number of stillborn, number of born per litter, number of weaned per litter

Oestrous cyclicity (parental animals):

Pregnancies were timed by vaginal smearing (Long and Evans, 1922)

Litter observations:

All newborns were counted at birth, but not handled until they were 4 days old. They were then weighed, sexed and inspected grossly.
- F1a: Discarded
- F1b: 5 per sex were necropsied and examined histologically.
- F1c: Teratologic evaluation (one-half of each treatment group on day 13, the other half on day 21 of gestation period). The pregnant females were examined for number of resorptions, corpora lutea and implantations
- F2a: Discarded
- F2b: Teratologic evaluation (one-half of each treatment group on day 13, the other half on day 21 of gestation period). The pregnant females were examined for number of resorptions, corpora lutea and implantations.

Postmortem examinations (parental animals):

Pregnant females were examined for number of resorptions, corpora lutea and implantations

Postmortem examinations (offspring):

Teratologic evaluation: Foetuses were examined for skeletal defects, soft-tissue defects, gross abnormalities

Statistics:

Analyses of variance were done on the appropriate data (Snedecor, 1946), and the partitioning was done by the Tukey "minimum difference" test as described by Scheffe (1952).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality was observed in four females, two were in the group administered 0.5% HEDP-2Na continuously and two were from the group receiving the high level during organogenesis. The cause of death could not be determined in two cases. However, death of the other two was attributed to pneumonia in one and to thyroid tumour in the other.
A female administered 0.5% HEDP-2Na during gestation, died on day 25 of the third phase, 2 days after giving birth to 7 dead pups. Five more pups were found in the uterus at necropsy. One of these was exencephalic and four were hydroencephalic; they could not be inspected further because of decomposition.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The weight gains were similar for all dose groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No differences were observed between treated and control animals.

Food efficiency:

no effects observed

Description (incidence and severity):

No differences were observed between treated and control animals.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No differences were observed in the growth between treated and control animals.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The overall conception rate of 90% shows that the compound did not interfere with implantation. Two litters, with an indeterminate number of pups, were stillborn in the first litters; one each in females administered 0.5 or 0.1% HEDP-2Na continuously. The former had a normal second litter, but the latter did not become pregnant again.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The overall conception rate of 90% shows that the compound did not interfere with the spermatogenesis.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

An increase in the number of stillborn foetuses of P1 litters were observed in dams administered 0.5% HEDP-2Na. Dams receiving 0.1% HEDP-2Na only during pregnancy had significantly more pups than either control rats or those administered the same level continuously. There were no significant differences in the numbers of resorptions or of implantations in females sacrificed at 13 days postcoitum; corpora lutea were not counted in these females.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

447 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed

Remarks on result:

other: equivalent to 368 mg/kg bw/day active acid

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

There was a significant reduction in the number of live pups born to dams administered 0.5% HEDP-2Na during organogenesis in the F1a phase and an increase in the number of stillborn foetuses, although these dams had a higher average number of pups than the control group.

In the P1 phase, those dams receiving 0.1% HEDP-2Na only during pregnancy had significantly more pups than either control rats or those administered the same level continuously. There were no significant differences in the preweaning mortality. Two litters, with an indeterminate number of pups, were stillborn in the first litters; one each in females administered 0.1 or 0.5 % HEDP-2Na continuously. The former had a normal second litter, but the latter did not become pregnant again. A female administered 0.5% HEDP-2Na during gestation, died on day 25 of the third phase, 2 days after giving birth to 7 dead pups. Five more pups were found in the uterus at necropsy. One of these was exencephalic and four were hydroencephalic; they could not be inspected further because of decomposition.

There was no significant mortality in rat embryos from the females sacrificed at 13 days in either generation.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The weight gains were similar for all dose groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No differences were observed between treated and control animals.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not specified

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Three malformed foetuses were found in 42 litters (0.6% incidence); 1 of these was from a female administered 0.1% HEDP-2Na continuously (left 12th rib missing) and 2 were from a female administered 0.5% of the test material during organogenesis (hydroencephalocele in 1, sternoschisis in the other). However, these changes were not considered as treatment-related.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No differences were observed in the growth between treated and control animals. There was a significant reduction in the number of live pups born to dams fed 0.5% of HEDP-2Na during organogenesis in the F1a phase although these dams had a higher average number of pups than the control mothers. The overall conception rate of 90% shows that the compound did not interfere with the spermatogenesis or nidation. The number of pups born and weaned were comparable to historical controls.

Teratologic evaluation of the third litters (F1c) showed no significant treatment differences in the number of live foetuses, corpora lutea, or implantations in females sacrificed at 21 days postcoitum. However, significantly more resorptions occurred in the control (untreated) group. In addition, 7 rats scattered among the test and control group resorbed all their embryos.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

447 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed

Remarks on result:

other: equivalent to 368 mg/kg bw/day active acid).

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

No significant mortality in rat embryos was observed from the females sacrificed at 13 days in either generation. However, in the second generation, there were fewer foetuses in the females administered 0.5% HEDP-2Na. Although varying statistical evidence, these collective data indicate that the 0.5% dose level of is somewhat toxic to the embryo and that it is slightly more toxic when provided only during pregnancy, rather than when administered chronically.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Second-generation foetuses were slightly heavier than those born in the first generation, however, the weights were not significantly different among treatment groups.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

The first litters of the second generation females were smaller than the previous generation, however, there were no significant differences in any of the parameters among the groups. Overall, 1.2% of the 1028 foetuses examined were defective, including 2.5% of the 211 foetuses from the control group. No defect was preponderant. Some variation was observed in the incidence of extra ribs and the number of sternebrae, however, the incidences of both were low and scattered randomly throughout both control and experimental groups.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

In the second litters used for teratologic evaluation, there were no significant differences observed among corpora lutea, implantations or resorptions at any time periods. At 21 days, the number of implantations was reduced in rats administered 0.5% HEDP-2Na continuously but was statistically different only from those administered 0.1% of the test substance during organogenesis. Corpora lutea formation in rats administered 0.5% HEDP-2Na continuously and sacrificed at 21 days was significantly depressed as compared with control animals. There was a decrease in the number of live foetuses born to both groups of dams receiving 0.5% of the test substance, however, the decrease was significant only in the group treated only during gestation.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

447 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Remarks on result:

other: equivalent to 368 mg/kg bw/day active acid.

Target system / organ toxicity (F2)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

TABLE 1: EFFECTS ON THE REPRODUCTIVE PERFORMANCE OF PARENT GENERATION (F0) RATS AND THEIR FIRST TWO LITTERS (F1a and F1b)

 

Group	No. pregnant		Conception rate		No. of stillborn		Av. No. born per litter		Av. No. weaned per litter		Av. weaning weight (g)
	F1a	F1b	F1a	F1b	F1a	F1b	F1a	F1b	F1a	F1b	F1a	F1b
Untreated control	17	19	85	95	2	6	13.0	10.4	7.6***	7.1***	47.6	46.9
0.5% continuously	18	17	90	89.5	2	9	12.6	12.8	7.9	7.5	47.3	44.3
0.1% continuously	18	16	90	80	4	10	12.2	10.2	7.8	7.0	44.8	47.4
0.5% day 6-15 postcoitum	19	18	95	94.7	5	22	9.8*	12.7	7.1	7.6	47.1	48.3
0.1% day 6-15 postcoitum	19	18	95	90	2	15	12.7	13.5**	7.5	7.9	46.9	44.8

 

* Significantly different from control rats (p<0.05)

** Significantly different from control rats and those fed 0.1% continuously (p<0.05)

*** All litters containing more than 8 pups were reduced to that number at 4 d

 

 

TABLE 2: EFFECTS ON THE REPRODUCTION OF PARENT GENERATION (F0) RATS AND ON THEIR THIRD LITTERS (F1C)

 

Effect	Control		0.5% continuously		0.1% continuously		0.5% days 6-15 postcoitum		0.1% days 6-15 postcoitum
	13 d	21 d	13 d	21 d	13 d	21 d	13 d	21 d	13 d	21 d
No. pregant	9	10	10	8*	9	9	9*	8*	9	7
% pregnant	90.0	100.0	100.0	88.9	90.0	90.0	100.0	88.9	90.0	70.0
No. total resorptions	1	2	1	0	1	0	1	0	1	0
Av. No. corpora lutea	-	15.3	-	14.0	-	14.3	-	12.8	-	15.5
Av. No. implantations	13.0	13.4	14.3	14.4	12.8	13.6	16.3	13.4	13.3	14.4
Av. No. resorptions	2.0	3.9**	0.3	0.0	1.3	0.0	1.8	0.0	0.6	0.0
Av. No. live fetuses	-	11.6	-	14.4	-	14.3	-	13.4	-	14.4
No. dead fetuses	-	0	-	0	-	0	-	1	-	0
No. fetuses abnormalities	-	0	-	0	-	1	-	2	-	0
Av. weight fetuses (g)	-	3.5	-	3.5	-	3.9	-	3.5	-	3.7

 

* Deaths earlier had reduced these groups in number

** Significantly different from other females sacrificed at 21 d (p<0.05)

 

 

TABLE 3: EFFECTS ON THE REPRODUCTION OF SECOND GENERATION PARENTS (F1b) AND ON THEIR FIRST LITTERS (F2a)

 

	Control	0.5% continuously	0.1% continuously	0.5% days 6-15 postcoitum	0.1% days 6-15 postcoitum
No. pregant	19	15	20	19	18
% pregnant	95.0	75.0	100.0	95.0	90.0
Av. No. born live per litter	10.2	9.1	8.9	9.1	10.3
Av. pup weight at 4d (g)	9.7	9.5	10.4	10.1	9.6
Av. No. weaned per litter*	7.1	7.4	6.7	7.0	7.4
Av. weight of pups  at weaning (g)	43.0	41.7	46.0	45.5	42.9

 

* Litters containing more than 8 pups were reduced to that number at 4 d

 

 

 

TABLE 4: EFFECTS ON THE REPRODUCTION OF SECOND GENERATION PARENTS (F1b) AND ON THEIR SECOND LITTERS (F2b)

 

Effect	Control		0.5% continuously		0.1% continuously		0.5% days 6-15 postcoitum		0.1% days 6-15 postcoitum
	13 d	21 d	13 d	21 d	13 d	21 d	13 d	21 d	13 d	21 d
No. pregant	10	10	10	7	10	9	9	9	10	10
% pregnant	100.0	100.0	100.0	70.0	100.0	90.0	90.0	90.0	100.0	100.0
No. total resorptions	0	1	1	0	1	0	2	1	1	0
Av. No. corpora lutea	13.9	14.2	13.6	11.9*	13.6	13.6	13.4	13.4	13.2	13.8
Av. No. implantations	14.3	13.1	12.0	10.7**	13.0	13.4	13.3	11.2	13.0	13.5
Av. No. resorptions	1.2	1.4	0.9	0.9	1.8	0.9	1.9	3.8	1.3	0.9
Av. No. live fetuses	-	13.0	-	9.9	-	12.4	-	9.1***	-	12.6
No. dead fetuses	-	0	-	0	-	0	-	0	-	0
No. fetuses abnormalities	-	5	-	1	-	1	-	0	-	3
Av. weight fetuses (g)	-	4.8	-	5.4	-	5.4	-	4.9	-	5.2

 

*Significantly less than control rats at 21 d (p<0.05)

** Significantly less than rats fed 0.1% days 6-15 postcoitum (p<0.05)

*** Significantly less than control rats (p<0.05)

 

 

TABLE 5: INCIDENCES AND DISTRIBUTION OF FETAL ABNORMALITIES OBSERVED IN RATS

 

	Control		0.5% continuously		0.1% continuously		0.5% days 6-15 postcoitum		0.1% days 6-15 postcoitum
	F1c	F2b	F1c	F2b	F1c	F2b	F1c	F2b	F1c	F2b
No. litters	7	9	8	7	9	9	7	8	7	10
No. fetuses examined	93	118	115	69	122	113	94	74	102	128
No. fetuses examined for skeletal defects	31	37	38	22	39	38	30	24	33	43
No. fetuses examined for soft-tissue defects	62	81	77	47	83	75	64	50	69	85
Fetuses with gross abnormalities (%)	0	2.5*	0	0	0	0	1.1	0	0	0
Fetuses with skeletal defects (%)	0	0	0	0	2.5~	0	3.3	0	0	0
Fetuses with soft-tissue defects (%)	0	5.0#	0	2.1	0	1.3	1.1	0	0	3.5
Facial dysgenesis	-	1.7	-	-	-	-	-	-	-	-
Hematoma	-	0.8	-	-	-	-	-	-	-	-
Hydronephrosis	-	2.5	-	-	-	1.3	-	-	-	1.2
Double aorta	-	1.2	-	-	-	-	-	-	-	-
Cryptorchism	-	-	-	2.1	-	-	-	-	-	-
Hydro-encephalocele	-	-	-	-	-	-	1.1	-	-	-
Ascites	-	-	-	-	-	-	-	-	-	1.2
Hyperplastic testicle	-	1.2	-	-	-	-	-	-	-	-
Abdominal hemorrhaging	-	-	-	-	-	-	-	-	-	1.2
Missing rib, L 12th	-	-	-	-	2.5	-	-	-	-	-
Sternoschisis	-	-	-	-	-	-	1.1	-	-	-

 

* Percentages are based upon total number of fetuses examined

~ Percentages are based upon number cleared and stained

# Percentages are based upon number of animals examined for soft-tissue effects

Applicant's summary and conclusion

Conclusions:

In a two-generation feeding study (reliability score 2), conducted to a protocol similar to OECD Test Guideline 416 and pre-GLP, HEDP (2-3Na) was administered to rats in doses of 0, 0.1 or 0.5 % (equivalent to 0 mg/kg bw/day, approximately 112 mg/kg bw/day and approximately 447 mg/kg bw/day respectively), either continuously to both sexes and to females only on Gestation Day 6-15. An impairment of reproductive function was not observed in male or female rats at relatively high dose levels. No treatment-related malformations or developmental variations were noted at any exposure level. Mean numbers of corpora lutea, implantation sites, resorptions, viable foetuses and mean foetal weights were unaffected by treatment at an exposure level of 112 mg/kg bw/day. In the second generation, mortality was observed at the highest dose tested. It was concluded that a NOAEL for maternal dams, the F1 and F2 generations are 447 mg/kg bw/day (equivalent to 368 mg/kg bw/day active acid).