Calcium cyanamide

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

other information

Study period:

1990

Reliability:

other: The reliability of the original study report and endpoint record is Klimisch 1, but as read-across for this endpoint is not supported, Klimisch rating is not applied. For justification against read-across see “Justification for type of information”.

Rationale for reliability incl. deficiencies:

other: Justification for reliability see “Justification for type of information”

Justification for type of information:

The study was conducted with cyanamide, which is considered an analogue substance to calcium cyanamide. However, for this specific endpoint, read-across from cyanamide to calcium cyanamide is not appropriate. The detailed justification against using read-across can be found in chapter 13.2 under the title “Scientific Rationale for not using Cyanamide as Read-Across Substance for Calcium Cyanamide on Toxicological Endpoints”. This endpoint study record is nevertheless copied to the calcium cyanamide dossier to demonstrate full consideration of all cyanamide study data in the substance assessment and classification of calcium cyanamide.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

(1981)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Version / remarks:

(1982)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Cyanamide

EC Number:

206-992-3

EC Name:

Cyanamide

Cas Number:

420-04-2

Molecular formula:

CH2N2

IUPAC Name:

cyanamide

impurity 1

Reference substance name:

Cyanoguanidine

EC Number:

207-312-8

EC Name:

Cyanoguanidine

Cas Number:

461-58-5

Molecular formula:

C2H4N4

IUPAC Name:

2-cyanoguanidine

additive 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Dihydrogen oxide

additive 2

Reference substance name:

Orthophosphoric acid

EC Number:

231-633-2

EC Name:

Orthophosphoric acid

Cas Number:

7664-38-2

Molecular formula:

H3O4P

IUPAC Name:

phosphoric acid

Test material form:

liquid - solid: mixture of

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

The rats were 7 week old.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

the dosing solutions were prepared weekly by dilution with distilled water and were stored under refrigeration. Approximately 1 hour prior to application the solutions were thawed and stirred during dosing. The application volume was 10 ml/kg bw.

Details on mating procedure:

One female was mated with one male overnight for a period of up to 3 weeks to produce the F1 litter. From the F1 pups 26 males and 26 females/dose group were selected as F1 parental generation to produce the F2 generation.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

14 weeks prior to mating, and dosing continued until termination.

Frequency of treatment:

Daily

Details on study schedule:

F0 males were dosed once daily for approximately 14 weeks prior to mating, and dosing continued until termination. F0 females were dosed once daily for approximately 14 weeks prior to mating and throughout the mating, pregnancy, lactation and post lactation periods. 1 female was mated with 1 male overnight for a period of up to 3 weeks to produce the F1 litter. From the F1 pups 26 males and 26 females/dose group were selected as F1 parental generation to produce the F2 generation following treatment with dose levels of 0, 1.25, 3.75 and 15.0 mg/kg bw/day for at least 14 weeks.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 5, 15 and 60 mg/kg bw/day (corresponding 0, 2.5, 7.5 and 30.0 mg/kg bw/day active ingredient) for 12 weeks. 0, 2.5, 7.5 and 30 mg/kg bw/day (corresponding 0, 1.25, 3.75 and 15.0 mg/kg bw/day pure active ingredient) from week 12 onwards.
Basis:
nominal conc.

No. of animals per sex per dose:

26 animals per sex per dose

Control animals:

yes, sham-exposed

Details on study design:

From the beginning of week 12 the doses were lowered due to severe impact on health expressed by loss in body weights of the F0 high dose animals.

Positive control:

No positive control

Examinations

Parental animals: Observations and examinations:

The examination of parental animals included monitoring for clinical symptoms/mortalities, food consumption, body weight development, mating and reproductive performances.

Litter observations:

All pups were examined macroscopically at necropsy (external and organ findings), stillborn pups and pups that died intercurrently were additionally examined for any skeletal findings.Litter size reduction in litters with more than 8 pups was performed on postnatal day 4.

Postmortem examinations (parental animals):

Pathological examination was performed by gross inspection as well as by histopathological examination with special attention to the organs of the reproductive system. Pups were sexed, weighed and monitored with respect to their viability and growth.

Postmortem examinations (offspring):

Culled pups were sacrificed by intraperitoneal injection of sodium pentobarbital and examined for visceral abnormalities.

Statistics:

A significant difference between the control and the treated groups was statistically examined by the Dunnett´s test (p < 0.05, p< 0.01).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

Signs of an effect in the high dose males were apparent after several weeks of treatment and included rough hair coats, thin appearance and substantially lower mean body weight gains compared to the control group. The body weight effect was also evident in the high dose females and mid dose males and females during the F0 growth phase. In these animals a lower mean food consumption tended to parallel the body weight effect in these animals. Thus, dose levels were reduced at week 12 of treatment.
After reduction of the dose levels towards the end of pre-mating, the body weight gain improved and especially the males of all treatment groups gained weight above the respective control during the post-mating period.
Mating ability was unimpaired by treatment in the F0 (and the F1 generation). Most females mated during the first oestrus and the distribution of matings that occurred at second oestrus or later did not indicate a test substance-related effect. A low fertility and gestation index were obtained in the F0 animals at the high dose group.
Gross pathological and histopathological data from F0 (and F1 adults) did not reveal any significant treatment-related changes.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

for maternal and developmental parameters

Effect level:

1.25 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: 1.25 mg/kg bw/day of the pure active ingredient cyanamide.

2

Dose descriptor:

NOAEL

Remarks:

for reproduction pararmeters

Effect level:

3.75 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: 3.75 mg/kg bw/day of the pure active ingredient cyanamide.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

Clinical observations of the F1 animals did not reveal any evidence of a treatment-related effect. Mean body weight gain values were lower for the high dose F1 males compared to the control animals for the during pre- and post-mating periods. In addition, an effect on body weight gain was also observed for the mid dose males during post-mating and high dose females during premating and gestation but not during lactation. Food consumption of the F1 animals showed lower values in the high dose group compared to the controls during the growth, gestation and lactation phases.
Mating ability was unimpaired by treatment in the F0 and the F1 generation. Most females mated during the first oestrus and the distribution of matings that occurred at second oestrus or later did not indicate a test substance-related effect. Low fertility and gestation indices were obtained in the F1 (and F0).
No substance-induced morphological abnormalities were found in the offspring. Neonatal survival (day 0 - 4) of the F1 and the F2 pups, however, was significantly lower in all treated groups compared to the control group. The investigators do not consider this to be treatment-related because of considerable variability in the historical data base. However, the historical data have not been provided for comparison and the reviewer considers the concurrent control to be more representative for the actual study conditions than historical control ranges.
F1 neonates had reduced birth weights in the mid and high dose group. Although the finding in the intermediate dose could be related to the higher mean live litter size the comparison with the low dose indicates that a test substance effect on foetal growth was present. Weaning weights were also lower in the mid and high dose groups. For the F2 pups at the high dose level a slight effect on birth weight was observed and the weights attained at weaning were marginally lower than in the control.
Birth weights of the F1 and F2 pups were not affected. In the F1 pup growth during lactation was retarded in the mid and high dose groups compared to the control group but not during raise of the F2 pups.
No treatment-related morphological alterations were observed in the F1 and F2 pups during inspection for cervical, thoracic and abdominal visceral abnormalities in the culled, intermittent died or scheduled killed pups.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Body weight gain data of F0/F1 parental animals:

Dose level (mg/kg bw/day)	0	2.5	7.5	30
F0 males - pre-mating (week 0-12)	271	249	224**	108**
F0 females
- pre-mating (week 0-12)	129	126	106**	59**
Dose level (mg/kg bw/day)	0	1.25	3.75	15
F0 males - post-mating (week 12-24)	68	92*	106*	99*
F0 females
- pregnancy (GD 0-20)	130	143	139	111
- lactation (PND 0-21)	-1.4	0.6	3.0	25
F 1 males - pre-mating (week 0-12)	461	446	441	391*
F 1 males - postmating (week 12-27)	111	101	87*	36*
F 1 females
- pre-mating (week 0-12)	221	232	222	202**
- pregnancy (GD 0-20)	119	129	126	94**
- lactation	1.4	-7.5	6.6	27**
* p < 0.05; ** P < 0.01 (Dunnett test)

Fertility and litter data of F0/F1 generation animals:

Dose level (mg/kg bw/day)	0	1.25	3.75	15
F0 generation
Cohabitated pairs	26	26	26	26
Mated females	26	22	24	23
Pregnant females	20	20	20	15*
Female fertility index (%)	77	91	83	65
Male fertility index (%)	80	91	83	65
Gestation index (%)	100	100	100	80
Mean duration of pregnancy (days)	22.06	22.11	21.89	22.10
Total prenatal litter loss	0	0	0	3
Live litters	20	20	20	12
Pups delivered (mean)	13.15	15.00	14.95	10.83
Live birth index (%)	98	96	96	99
Viability index (alive PND 0-4)	92	83**	88**	84**
(HCD: 94.7%±5.6, Min.: 84% - Max.: 98%, supplied by breeder)
(HCD: % dead pups prior to culling: 2.76% ±2.76, Min.: 0.0% - Max.: 13.1% supplied by MARTA, 1996)
Weaning index (alive PND 4–21)	92	85	87	88
(HCD: 95.2%±10.9, Min.: 73% - Max.: 100%, supplied by breeder)
(HCD: % dead pups post-culling - weaning: 0.71% ±1.28, Min.: 0.0% - Max.: 8.6% supplied by MARTA, 1996)
Male pup weight PND 0 (g)	6.70	6.59	6.49	6.28
Female pup weight PND 0 (g)	6.34	6.27	6.05	5.89
Male pup weight PND 21(g)	50.48	47.13	43.20*	42.62*
Female pup weight PND 21 (g)	49.04	47.03	40.43**	42.26*
F1 generation
Cohabitated pairs	26	26	26	26
Mated females	25	24	26	23
Pregnant females	24	21	23	19
Female fertility index (%)	96	88	88	83
Male fertility index (%)	96	88	88	83
Gestation index (%)	96	95	91	89
Mean duration of pregnancy (days)	21.8	22.0	21.9	22.0
Total prenatal litter loss	0	0	2	2
Live litters	23	20	21	17
Pups delivered (mean)	12.87	13.45	15.05*	12.29
Live birth index (%)	98	98	98	95
Viability index (alive PND 0-4)	93	87*	82**	81**
(HCD: 94.7%±5.6, Min.: 84% - Max.: 98%, supplied by breeder)
(HCD: % dead pups prior to culling: 2.76% ±2.76, Min.: 0.0% - Max.: 13.1% supplied by MARTA, 1996)
Weaning index (alive PND 4–21)	87	81	87	97*
(HCD: 95.2%±10.9, Min.: 73% - Max.: 100%, supplied by breeder)
(HCD: % dead pups postculling - weaning: 0.71% ±1.28, Min.: 0.0% - Max.: 8.6% supplied by MARTA, 1996)
Male pup weight PND 0 (g)	6.42	6.57	6.58	6.20
Female pup weight PND 0 (g)	6.02	6.14	6.24	5.83
Male pup weight PND 21 (g)	44.77	43.11	43.78	42.65
Female pup weight PND 21 (g)	43.22	42.68	41.13	39.99

It should be taken in account that due to the change of dose levels just before mating of the F0 animals, the apparent effects on fertility and neonatal growth and survival both in the highest dose group were attributed to treatment but cannot be attributed to a specific dose level. The general health of these animals was influenced and not comparable to that of the control animals at the time of mating and the reproductive effects occurred in the presence of a clear maternal effect.

Applicant's summary and conclusion

Conclusions:

The following findings were obtained in study: Clinical findings, reduced body weight and food consumption in parental animals of mid and high dose (F0 and F1 generation).
Reduced fertility at high dose.Retarded body weight gain (F1 pups) and reduced survival (F2 pups) in mid and high dose group.No morphological alterations.
Thus, NOAEL of 1.25 mg/kg bw/day pure active ingredient cyanamide was determied for maternal and developmental parameters and NOAEL of 3.75 mg/kg bw/day pure active ingredient was determied for reproduction parameters

Executive summary:

The continuous application of aqueous hydrogen cyanamide to Sprague-Dawley rats over two generations at dose levels of 0, 2.5, 7.5 and 30 mg/kg bw/day pure active ingredient during the first 12 weeks of treatment followed by 0, 1.25, 3.75 and 15 mg/kg bw/day of pure active ingredient orally by gavage affected reproductive performance and fertility of the F0 or F1 parental animals only in form of reduced fertility indices in F0 and F1 parental animals at the top dose group of 30/15 mg/kg bw/day active ingredient. All other observable differences between both of these groups and the concurrent controls were regarded to be incidental in nature and not of toxicological or biological concern.

Signs of general, systemic toxicity in both parental generations (F0 and F1) were confined to the rats of the mid and high dose levels. Toxicity was characterized by effects on food consumption and body weights and body weight gain during premating within the first 12 weeks. After reduction of the dose levels prior to mating of the F0 parental animals, the health status improved continuously. However, an impairment of body weight gain was also noted for the F1 parental males at the mid and high dose level (i.e. e. 3.75 and 15 mg/kg bw/day active ingredient) especially in the post-mating period and for the high dose F1 males during pre- and post-mating as well as for the high dose F1 females during premating and gestation. Concerning pathology, none of the recorded gross lesions or microscopic findings in the F0 and F1 generation parental animals was interpreted to represent an adverse treatment related effect.

Substance induced signs of developmental toxicity were observed in the progeny of the F0 parental generation in form of retarded body weight gain and in the F2 pups in form of reduced survival during early postnatal live resulting in decreased viability indices in the mid and high dose groups. In none of the F1 or F2 pups substance-induced morphological alterations were noted by external and visceral inspection.

Thus, under the conditions of this study the NOAEL (no observed adverse effect level) for reproductive performance and fertility is 3.75 mg/kg bw/day pure active ingredient due to the reduced fertility indices in both generations. The NOAEL for parental and developmental toxicity is considered to be 1.25 mg/kg bw/day pure active ingredient.