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Key value for chemical safety assessment

Effects on fertility

Description of key information

Lowest NOAEL for female is 4.5 mg/kg bw/day (oral, rat) = 75 mg/kg diet ( Birth weight of the offspring)

Lowest NOAEL for male is 300 mg/kg diet (decrease in properly motile spermatozoa (live) number in the parental and F1 generation)

(15,73 mg TNT/kg of feed/24h in case of parental generation and 26,94 mg TNT/kg body weight/24h in case of second generation F1)

The classification as Repr. 2, H361f is regarded as inappropriate since:

- No statistically significant effect on female oestrous cyclicity or estrogen cycle length was found

- No pathological pregnancy was observed, no abnormalities during delivery or increased neonatal deaths during delivery, feeding and adolescence

- No abnormalities in the structure of the gonads and their microscopic image were observed in both females and males of parental and second generations. There were no signs of degenerative, inflammatory, cancerous changes within the reproductive organs that could affect their ability to reproduce

- A significant reduction in the number of correctly motile spermatozoa was found in the 1200 mg TNT/kg feed males dosed in parental generation by 11.51%. Despite the decrease in this parameter, the sperm quality was sufficient for 100% fertilization efficiency in the group.

- The test item did not affect the length of pregnancy.

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Deviations:
yes
Remarks:
The test parameters: 19-25°C, relative humidity 30-70%. During the experiment: lowest temp. 18.70°C; highest 25.10°C; highest humidity 71%. These were short-term deviations and did not affect the course of the study or the reliability of the results.
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
oral: feed
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg diet
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Remarks on result:
other: 4,50 mg TNT/kg body weight/24h – average dose for mothers in pregnancy period
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg diet
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive function (sperm measures)
Remarks on result:
other: 15,73 mg TNT/kg of feed/24h in case of parental generation and 26,94 mg TNT/kg body weight/24h in case of second generation F1
Dose descriptor:
NOAEL
Effect level:
300 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: 15,73 mg TNT/kg body weight/24h – males; 22,94 mg TNT/kg body weight/24h – females
Dose descriptor:
NOAEL
Effect level:
300 mg/kg diet
Based on:
test mat.
Sex:
male
Basis for effect level:
food consumption and compound intake
Remarks on result:
other: 15,73 mg TNT/kg body weight/24h
Dose descriptor:
NOAEL
Effect level:
< 75 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1A)
Effect level:
300 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: 26,94 mg TNT/kg body weight/24h – males; 26,15 mg TNT/kg body weight/24 h - females
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1B)
Effect level:
< 75 mg/kg diet
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: <7,14 mg TNT/kg body weight/24h
Dose descriptor:
NOAEL
Generation:
F1 (cohort 2A)
Effect level:
< 75 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: 7,72 mg TNT/kg body weight/24h – males; 7,14 mg TNT/kg body weight /24 h – females
Dose descriptor:
NOAEL
Generation:
F1 (cohort 2B)
Effect level:
300 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: 26,94 mg TNT/kg body weight/24h – males; 26,15 mg TNT/kg body weight/24 h – females
Reproductive effects observed:
not specified

NOAEL and LOAEL values were defined for parameters:

- Amount of feed consumed by parental generation in male group:

NOAEL – 300 mg TNT/kg of feed (15,73 mg TNT/kg body weight/24h)

LOAEL – 1200 mg TNT/kg of feed (70,09 mg TNT/kg body weight/24h).

- Body weight of parental generation animals in female and male group:

NOAEL – 300 mg TNT/kg of feed (15,73 mg TNT/kg body weight/24h – males; 22,94 mg TNT/kg body weight/24h – females)

LOAEL – 1200 mg TNT/kg of feed (70,09 mg TNT/kg body weight/24h – males; 81,96 mg TNT/kg body weight/24h – females).

- Body weight of F generation, cohort 1A in female and male group:

NOAEL – 300 mg TNT/kg of feed (26,94 mg TNT/kg body weight/24h – males; 26,15 mg TNT/kg body weight/24 h - females)

LOAEL – 1200 mg TNT/kg of feed (105,63 mg TNT/kg body weight/24h – males; 101,85 mg TNT/kg body weight/24 h - females).

- Body weight of F generation, cohort 1B in females and male group:

NOAEL < 75 mg TNT/kg of feed (<7,14 mg TNT/kg body weight/24h)

LOAEL < 75 mg TNT/kg of feed (7,14 mg TNT/kg body weight/24h) in case of females

NOAEL – 75 mg TNT/kg of feed (7,72 mg TNT/kg body weight/24h)

LOAEL – 300 mg TNT/kg of feed (26,94 mg TNT/kg body weight/24h) in case of males.

- Body weight of F generation, cohort 2A in female and male group:

NOAEL, LOAEL < 75 mg TNT/kg of feed (7,72 mg TNT/kg body weight/24h – males; 7,14 mg TNT/kg body weight /24 h – females).

- Body weight of F generation, cohort 2B in female and male group:

NOAEL – 300 mg TNT/kg of feed (26,94 mg TNT/kg body weight/24h – males; 26,15 mg TNT/kg body weight/24 h – females).

LOAEL – 1200 mg TNT/kg of feed (105,63 mg TNT/kg body weight/24h – males; 101,85 mg TNT/kg body weight/24 h – females.

- Birth weight of the offspring:

NOAEL – 75 mg TNT/kg of feed (4,50 mg TNT/kg body weight/24h – average dose for mothers in pregnancy period) in case of females

LOAEL – 300 mg TNT/kg of feed (16,33 mg TNT/kg body weight/24h – average dose for mothers in pregnancy period) in case of females

NOAEL – 300 mg TNT/kg of feed (16,33 mg TNT/kg body weight/24h – average dose for mothers in pregnancy period) in case of males mothers in pregnancy period) in case of females

LOAEL – 1200 mg TNT/kg of feed (61,20 mg TNT/kg body weight/24h – average dose for mothers in pregnancy period) in case of males.

- Sperm parameters – decrease in properly motile spermatozoa (live) number in the parental and F1 generation:

NOAEL – 300 mg TNT/kg of feed (15,73 mg TNT/kg of feed/24h in case of parental generation and 26,94 mg TNT/kg body weight/24h in case of second generation F1)

LOAEL – 1200 mg TNT/kg of feed (70,09 mg TNT/kg of feed/24h in case of parental generation and 105,63 mg TNT/kg body weight/24h in case of second generation F1).

- Forelimb grip strength:

NOAEL – 300 mg TNT/kg of feed (26,94 mg TNT/kg body weight/24h – males; 26,15 mg TNT/kg body weight/24 h – females)

LOAEL – 1200 mg TNT/kg of feed (105,63 mg TNT/kg body weight/24h – males; 101,85 mg TNT/kg body weight/24 h – females).

- Number of erythrocytes, females and males parental generation:

NOAEL, LOAEL < 75 mg TNT/kg of feed (<4,51 mg TNT/kg body weight/24h – males; <6,74 mg TNT/kg body weight/24h – females).

- Number of erythrocytes, females and males F1 generation, cohort 1A:

NOAEL – 75 mg TNT/kg of feed (7,14 mg TNT/kg body weight/24h) in case of females

LOAEL – 300 mg TNT/kg of feed (26,15 mg TNT/kg body weight/24h) in case of females

NOAEL – 300 mg TNT/kg of feed (26,94 mg TNT/kg body weight/24h) in case of males

LOAEL – 1200 mg TNT/kg of feed (105,63 mg TNT/kg body weight/24h) in case of males.

- Haemoglobin concentration, females and males parental generation:

NOAEL, LOAEL < 75 mg TNT/kg of feed (<4,51 mg TNT/kg body weight/24h – males; <6,74 mg TNT/kg body weight/24h – females).

- Haemoglobin concentration, females and males cohort 1A, F1 generation:

NOAEL – 300 mg TNT/kg of feed (26,15 mg TNT/kg body weight/24h) in case of females

LOAEL – 1200 mg TNT/kg of feed (101,85 mg TNT/kg body weight in case of females

NOAEL – 75 mg TNT/kg of feed (7,72 mg TNT/kg body weight/24h) in case of males

LOAEL – 300 mg TNT/kg of feed (26,94 mg TNT/kg body weight) in case of males.

- Haematocrit, males and females parental generation:

NOAEL, LOAEL < 75 mg TNT/kg of feed (<4,51 mg TNT/kg body weight/24h – males; <12,15 mg TNT/kg body weight/24h – females).

- Haematocrit, females and males cohort 1A, F1 generation:

NOAEL – 300 mg TNT/kg of feed (26,94 mg TNT/kg body weight/24h)

LOAEL – 1200 mg TNT/kg of feed (105,63 mg TNT/kg body weight/24h) in case of males – no dose dependence for females.

- Average haemoglobin concentration in erythrocytes (MCHC) in case of F1 generation males:

NOAEL – 75 mg TNT/kg of feed (7,72 mg TNT/kg body weight/24h)

LOAEL – 300 mg TNT/kg of feed (26,94 mg/kg body weight).

- Number of granulocytes (decrease):

NOAEL – 75 mg TNT/kg of feed (4,51 mg TNT/kg body weight/24h) in case of males of parental generation

LOAEL – 300 mg TNT/kg of feed (15,73 mg TNT/kg body weight/24h) in case of males of parental generation

NOAEL – 300 mg TNT/kg of feed (26,15 mg TNT/kg body weight/24h) in case of females of F1 generation, cohort 1A

LOAEL – 1200 mg TNT/kg of feed (101,85 mg TNT/kg body weight/24h) in case of females of F1 generation, cohort 1A.

- Number of granulocytes with segmented nuclei (decrease):

NOAEL, LOAEL < 75 mg TNT/kg of feed (<4,51 mg/kg body weight /24h) in case of males of parental generation

NOAEL – 300 mg TNT/kg of feed (26,15 mg TNT/kg body weight/24h) in case of females of F1 generation, cohort 1A

LOAEL – 1200 mg TNT/kg of feed (101,85 mg/kg body weight /24h) in case of females of F1 generation, cohort 1A.

- Activated partial thromboplastin time (APTT) parental generation:

NOAEL, LOAEL < 75 mg TNT/kg of feed (<4,51 mg TNT/kg body weight/24h – males; <6,74 mg TNT/kg body weight/24h – females).

- Alanine aminotransferase (activity decrease) in case of females of parental generation:

NOAEL – 75 mg TNT/kg of feed (6,74 mg TNT/kg body weight/24h)

LOAEL – 300 mg TNT/kg of feed (22,94 mg TNT/kg body weight/24h).

- Aspartic aminotransferase (activity decrease) for cohort 1A males and females of F1 generation:

NOAEL – 300 mg TNT/kg of feed (26,94 mg TNT/kg body weight/24h – males; 26,15 mg TNT/kg body weight/24 h – females)

LOAEL – 1200 mg TNT/kg of feed (105,63 mg TNT/kg body weight/24h – males; 101,85 mg TNT/kg body weight/24 h – females).

- Globulins (concentration decrease) in F1 generation males, cohort 1A:

NOAEL – 300 mg TNT/kg of feed (26,94 mg TNT/kg body weight/24h)

LOAEL – 1200 mg TNT/kg of feed (105,63 mg TNT/kg body weight/24h).

- Cholesterol (concentration decrease) in parental generation males:

NOAEL, LOAEL < 75 mg TNT/kg of feed (<4,51 mg TNT/kg body weight/24h)

- Creatinine (concentration decrease) in F1 generation males, cohort 1A:

NOAEL – 75 mg TNT/kg of feed (7,72 mg TNT/kg body weight/24h)

LOAEL – 300 mg TNT/kg of feed (26,94 mg TNT/kg body weight/24h).

- Sodium – concentration of sodium decrease: NOAEL – 75 mg TNT/kg feed – females F1 generation, cohort 1A:

NOAEL – 75 mg TNT/kg of feed (7,14 mg TNT/kg body weight/24h)

LOAEL – 300 mg TNT/kg of feed (26,15 mg TNT/kg body weight/24h).

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
4.5 mg/kg bw/day
Species:
rat
Quality of whole database:
Klimisch rating 1.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

Pre-natal developmental toxicity study OECD 414 in a first species (rat) was finished in May 2018. Afterwards expert opinion prepared by a toxicologist was prepared to be sure that a second study is necessary.

As far as Pre-natal developmental toxicity study OECD 414 in a first species, classification of 2,4,6-trinitrotoluene as suspected human reproductive toxicant, category 2 (H361d: Suspected of damaging the unborn child) is regarded appropriate. This classification accommodates for both the positive findings in laboratory animals and the absence of significant effects in humans.

Since the substance does not meet the criteria for classification in category 1 of reproductive toxicity, the pre-natal developmental toxicity study (test method: EU 8.31./OECD TG 414) with rabbits as a second species by the oral route should be performed.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Oral route, rat or rabbit
Species:
rat
Strain:
Wistar
Route of administration:
oral: gavage
Key result
Dose descriptor:
NOAEL
Effect level:
45 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: na
Dose descriptor:
LOAEL
Effect level:
160 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
organ weights and organ / body weight ratios
Dose descriptor:
LOAEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
not specified
Developmental effects observed:
yes
Lowest effective dose / conc.:
10 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

- reduction in feed intake (statistically significant decrease compared to the control group) and water was observed at the exposure level of 160 mg/kg b.w. and 250 mg/kg b.w.

- maternal weight reduction and increase of methaemoglobin in a statistically significant differences were observed at the 160 mg/kg b.w. and 250 mg/kg b.w. dose levels.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
45 mg/kg bw/day
Species:
rat
Quality of whole database:
Klimisch rating 1.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

As far as Pre-natal developmental toxicity study OECD 414 in a first species, classification of 2,4,6-trinitrotoluene as suspected human reproductive toxicant, category 2 (H361d: Suspected of damaging the unborn child) is regarded appropriate. This classification accommodates for both the positive findings in laboratory animals and the absence of significant effects in humans.

Since the substance does not meet the criteria for classification in category 1 of reproductive toxicity, the pre-natal developmental toxicity study (test method: EU 8.31./OECD TG 414) with rabbits as a second species by the oral route should be performed.

The classification as Repr. 2, H361f is regarded as inappropriate since:

- No statistically significant effect on female oestrous cyclicity or estrogen cycle length was found

- No pathological pregnancy was observed, no abnormalities during delivery or increased neonatal deaths during delivery, feeding and adolescence

- No abnormalities in the structure of the gonads and their microscopic image were observed in both females and males of parental and second generations. There were no signs of degenerative, inflammatory, cancerous changes within the reproductive organs that could affect their ability to reproduce

- A significant reduction in the number of correctly motile spermatozoa was found in the 1200 mg TNT/kg feed males dosed in parental generation by 11.51%. Despite the decrease in this parameter, the sperm quality was sufficient for 100% fertilization efficiency in the group.

- The test item did not affect the length of pregnancy.

Additional information