Cyanamide

Endocrine disrupter testing in aquatic vertebrates – in vivo

Administrative data

Endpoint:

amphibian: other

Remarks:

Amphibian metamorphosis assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 May 2021 to 03 June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: U.S. EPA OPPTS Number 890.1100

Version / remarks:

2009

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD TG 231 (The Amphibian Metamorphosis Assay)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Nominal 0.048, 0.24, 1.2 and 6.0 mg a.s./L
- Sampling: Water samples were collected from each replicate test chamber two days prior to the start of the exposure period to confirm the concentrations after conditioning the diluter system. Water samples also were collected from each replicate test chamber at the beginning of the exposure, weekly during the exposure and at the end of the exposure period (Days 0, 8, 14 and 21) to measure concentrations of the test substance. An additional set of samples were collected from the negative control on Day 15.
A sample of the stock solution was collected for analysis prior to the exposure period to confirm the concentrations being delivered to the diluter system.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An aqueous stock solution was prepared weekly during the test. All test solutions were adjusted to 100 % active ingredient during preparation, based on the reported test substance purity (50.5 %). At each preparation, a single 350 mg a.s./L aqueous stock solution was prepared by mixing a calculated amount of test substance in 75-L of UV sterilized well water for approximately five hours in a 175 L stainless steel drum. The drum was mixed with a large top-down electric mixer. After preparation, the stock solution appeared clear and colorless. The stock solution was pumped into the diluter mixing chambers assigned to the treatment groups at target rates and was mixed with well water in the mixing chambers. The test solutions in the mixing chambers were stirred continuously and were protected from light. The test solutions were pumped from the mixing chambers into the test chambers at a target rate of 66 mL/min.
- Controls: The negative control received dilution water only.
- Test concentration separation factor: 5
- Evidence of undissolved material: no, the test solutions in both the mixing chambers and test chambers appeared clear and colorless, with no visible precipitates

Test organisms

Aquatic vertebrate type:

frog

Test organisms (species):

Xenopus laevis

Details on test organisms:

TEST ORGANISM
- Common name: African clawed frog
- Source: Eurofins Cultures, Easton, Maryland
- Life stage: tadpoles
- Age at study initiation: NF Stage 51

ACCLIMATION
- Acclimation period: yes, duration not specified
- Acclimation conditions: same as test
- Type and amount of food during acclimation: same as test
- Feeding frequency: two to three times per day
- Health during acclimation: healthy

FEEDING DURING TEST
- Food type: Sera Micron® (Sera North America, Montgomeryville, PA)
- Amount during main test: 15 to 40 mg Sera Micron®/animal/day, depending on study day
- Frequency: three times daily during the test and twice on the last day of the test

Environmental conditions
- photoperiod: 12 hours of light and 12 hours of darkness; a 30 minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

Hardness:

140 - 180 mg/L as CaCO3

Test temperature:

21.9 - 22.6 °C

pH:

7.7 - 8.1

Dissolved oxygen:

7.4 - 8.7 mg O2/L

Conductivity:

346 - 425 µS/cm

Nominal and measured concentrations:

Nominal: 0, 0.048, 0.24, 1.2 and 6.0 mg a.s./L
Mean measured: 0, 0.050, 0.25, 1.2 and 6.0 mg a.s./L

Details on test conditions:

TEST SYSTEM
- Test chamber: were 12 L glass aquaria filled with 10 L of test solution
- Type of flow-through: diluter system
- Renewal rate of test solution: The flow of the test solution through the peristaltic pumps was adjusted to provide approximately 10 volume additions of test solution in each test chamber per day
- No. of organisms per test chamber: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

OTHER TEST CONDITIONS
- Photoperiod: 12 hours of light and 12 hours of darkness; a 30 minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting
- Light intensity: 831 to 1346 lux (at the water surface)

EFFECT PARAMETERS MEASURED:
- Survival, gross morphological abnormalities, development stage, wet weight, snout-to-vent length, normalized hind-limb length, and thyroid histopathology

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 5

RANGE FINDING STUDY
- Test concentrations: 0.081, 2.7. 9.0, 30 and 100 mg a.s./L
- Results used to determine the conditions for the definitive study: yes

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: survival, developmental stage

Effect concentrations 2

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: wet weight, snout-to-vent length, normalized hind-limb lengths, histopathologic findings

Details on results:

- Survival: Tadpole survival to Day 7 in the control and all treatment groups was 100%. Tadpole survival to Day 21 in the negative control, 0.050, 0.25, 1.2 and 6.0 mg a.s./L treatment groups was 100, 100, 100, 100 and 98.8%, respectively. There were no statistically significant or treatment related effects on survival in any treatment group in comparison to the control on Day 21 (p > 0.05).
- Behaviour/abnormalities: Tadpoles in the control and all treatment groups generally appeared normal throughout the test. At termination, tail curvature was present in 38, 12, 7, 7 and 2 % of the tadpoles in the control, 0.050, 0.25, 1.2 and 6.0 mg a.s./L treatment groups, respectively. In feeding trials it has been shown that feeding rates during acclimation contribute to the amount of curvature observed. Therefore, the tail curvature was not considered to be a thyroid-related effect, but rather a dietary effect.
- Growth: There were 13 tadpoles greater than stage 60 at test termination that were excluded from statistical analysis: 2 tadpoles at stage 61 in the negative control, 1 tadpole at stage 61 and 3 tadpoles at stage 62 in the 0.050 mg a.s./L treatment group, 2 tadpoles at stage 61 and stage 62 in the 0.25 mg a.s./L treatment group and 3 tadpoles at stage 61 in
the 1.2 mg a.s./L treatment group.
- Wet weight: Mean wet weights on Day 7 in the control, 0.050, 0.25, 1.2 and 6.0 mg a.s./L treatment groups were 318, 287, 299, 291 and 228 mg, respectively. There was a statistically significant decreasing trend in Day 7 wet weight in the 6.0 mg a.s./L treatment group according to Jonckheere Terpstra trend test (p ≤ 0.05). Mean wet weights on Day 21 in the control, 0.050, 0.25, 1.2 and 6.0 mg a.s./L treatment groups were 1045, 1067, 1063, 1081 and 669 mg, respectively. There was a statistically significant decrease in Day 21 wet weights in the 6.0 mg a.s./L treatment group according to Dunnett’s test (p ≤ 0.05).
- Snout-to-Vent Length: Mean Day 7 snout-to-vent lengths in the control, 0.050, 0.25, 1.2 and 6.0 mg a.s./L treatment groups were 16.1, 15.5, 15.8, 15.4 and 14.2 mm, respectively. There was a statistically significant decreasing trend in Day 7 snout-to-vent length in the 6.0 mg a.s./L treatment group according to the Jonckheere-Terpstra trend test (p ≤ 0.05). Mean snout-to-vent lengths on Day 21 in the control, 0.050, 0.25, 1.2 and 6.0 mg a.s./L treatment groups were 24.2, 24.2, 24.2, 24.4 and 20.7 mm, respectively. There was a statistically significant decrease in Day 21 snout-to-vent length in the 6.0 mg a.s./L treatment group according to Dunnett’s test (p ≤ 0.05).
- Normalized Hind-Limb Length: Mean Day 7 normalized hind-limb lengths in the control, 0.050, 0.25, 1.2 and 6.0 mg a.s./L treatment groups were 0.20, 0.17, 0.18, 0.18 and 0.17 mm, respectively. There was a statistically significant decrease in Day 7 normalized hind-limb length in the 6.0 mg a.s./L treatment group according to the Jonckheere-Terpstra trend test (p ≤ 0.05). However, the difference between the lowest treatment group (0.172 mm) and the highest treatment group (0.165 mm) is very small (~4 %). Mean Day 21 normalized hind-limb lengths in the control, 0.050, 0.25, 1.2 and 6.0 mg a.s./L treatment groups were 0.43, 0.44, 0.50, 0.46 and 0.39 mm, respectively. There was a statistically significant increase in normalized hind-limb lengths in the 0.25 mg a.s./L treatment groups according to Dunnett’s test (p ≤ 0.05).
Upon further evaluation of the data, the increase in the 0.25 mg a.s./L treatment group was due to one replicate. In Replicate C there were 7 out of 15 tadpoles at NF stage 60, resulting in an increase in normalized hind-limb lengths. When this replicate was excluded from analysis for normalized hind-limb length, there was no statistical difference in normalized hind-limb lengths. In addition, this increase was not dose-responsive and falls within laboratory historical negative control data (0.45 ± 0.05 mm; range 0.40 – 0.54 mm; N = 19). This increase in the 0.25 mg a.s./L treatment group is likely attributed to biological variability and is not considered to be a treatment-related effect. This is confirmed by the statistical analysis of normalized HLL by NF stage. Statistical analyses of the normalized HLL separately for NF stages 57, 58, 59 and 60 reveals no statistically significant difference in normalized HLL to the control.
- Developmental Stage: There were no statistically significant effects on developmental stage distribution on Day 7 and at test termination on Day 21. The median developmental stage of the tadpoles on Day 7 was 53 for the control and all treatment groups. The median developmental stage of the tadpoles on Day 21 in the control, 0.050, 0.25, 1.2 and 6.0 mg a.s./L treatment groups were 57, 58, 58, 58 and 57, respectively.
- Histopatholoy: The histopathologic semi-quantitative and qualitative evaluation of 4 groups of Xenopus laevis tadpoles resulted in treatment-related findings in the 1.2 and 6.0 mg a.s./L treatment groups. Findings attributable to cyanamid exposure in these two groups consisted of an increased prevalence and severity of follicular cell hyperplasia, an increased prevalence (and severity in the 6.0 mg a.s./L treatment group) of follicular cell hypertrophy, and the occurrence of thyroid hypertrophy. While such effects are typically indicative of TSH stimulation of the thyroid gland, that conclusion is inconsistent with the lack of significant effects on median developmental stage and mean normalized hind-limb length. Meanwhile, the recorded decreases in mean wet weight and mean snout-to-vent length typically represent growth inhibition as opposed to thyroid-related effects on metamorphic development.

Reported statistics and error estimates:

Statistical analyses were performed to evaluate differences between treatment and control groups for each of the following biological endpoints:
• Survival
• Developmental Stage
• Body Weight
• Snout-to-Vent Length
• Hind-Limb Length (normalized by dividing by snout-to-vent length)
(Tadpoles > NF stage 60 excluded from growth analysis per guideline recommendations)
Unless otherwise noted, the unit of statistical analysis was the replicate test chamber. All data, except survival and developmental stage, was visually evaluated for monotonicity. If responses appeared to be monotonic (trending in one direction, e.g., response not trending up and then down as concentration increases) a step-down Jonckheere-Terpstra trend test was used to evaluate trends in the ranks of replicate Data for endpoints that were visually determined to be non-monotonic were analyzed by Dunnett’s test, and evaluated for normality using Shapiro Wilk’s test and for homogeneity of variance using Levene’s test (α = 0.01). All Day 7 growth data was visually determined to be monotonic, and all Day 21 growth data was visually determined to be non-monotonic.
Survival and developmental stage were not amenable to the statistical methods used for analysis of other endpoints. In particular, the most suitable unit of statistical analysis for these endpoints was the individual animal. Therefore, survival was analyzed using Fishers Exact test. In accordance with recommendations of OECD TG 231 and OPPTS 890.1100, analysis of metamorphic stage was performed using the multi-quantile analysis developed by T. Springer and J. Green, and using the step-down Jonckheere-Terpstra trend test on stage quantiles in the replicate test chambers.
All endpoints except for survival were evaluated using a two-tailed statistical test. Statistical tests used to evaluate treatment effects were performed at confidence level of α = 0.05 with SAS software.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In line with OECD 231, it is concluded that the observed effects in the amphibian metamorphosis test on wet weight, snout-to-vent length, HLL and histopathology are caused by general toxicity of the test item.
Considering the assessment scheme in OECD 231 (Figure 3 in the TG) it is concluded that the test item does not affect the thyroid as no test item related advanced development, no asynchronous development and no correlations to histological findings on the thyroid were observed.

Executive summary:

African clawed frog (Xenopus laevis) tadpoles were exposed to cyanamid at mean measured concentrations of 0.048, 0.24, 1.2 and 6.0 mg a.s./L for 21 days. The endpoints evaluated to determine if the test substance might impact the hypothalamus-pituitary-thyroid (HPT) axis of tadpoles were survival, developmental stage, wet weight, snout-to-vent length, normalized hind-limb length and thyroid gland histopathology.

There were no treatment-related effects on survival or developmental stage on Day 7 or Day 21. There was a statistically significant decrease on wet weights, snout-to-vent lengths and normalized hind-limb lengths in the 6.0 mg a.s./L treatment group on Day 7.

There were no treatment-related effects on survival, developmental stage or normalized hind-limb lengths on Day 21. There were statistically significant decreases in wet weight and snout-to-vent lengths in the 6.0 mg a.s./L treatment group on Day 21.

Histopathologic findings in the 1.2 and 6.0 mg a.s./L included increased prevalence and severity of follicular cell hyperplasia, an increased prevalence (and severity in the 6.0 mg a.s./L treatment group) of follicular cell hypertrophy, and the occurrence of thyroid hypertrophy. Although these findings are indicative of a thyroid effect, there were no effects on developmental stage or normalized hind-limb length in any treatment group at termination that would be consistent with the histopathological findings.

Therefore, under consideration of the assessment scheme presented in OECD 231, it is concluded that cyanamid has no potential to interfere with the hypothalamus-pituitary-thyroid axis of vertebrates.