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Toxicological information

Toxicity to reproduction

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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

1
Chemical structure
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 / concentrationsopen allclose all
Dose / conc.:
0.1 other: %
Remarks:
nominal in diet; equivalent to 112 mg/kg bw/day
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).