Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Reproductive toxicity as well as the general systemic toxic potential of DNAN were assessed (2018, Envigo, BC12JY). The study was conducted in accordance with OECD 421 and in compliance with GLP. The animals received doses of DNAN at 1, 5 and 20 mg/kg/day for two weeks prior to mating and up to the day before sacrificing inclusive (males) or up to days 13-15 of lactation (females) which were well tolerated. There were no effects on estrous cycle, pre-coital interval, mating performance, fertility and gestation length.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 November, 2017 - 22 August, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Physical form / color Pale yellow/solid
Batch number 55503625
Lot number 02/13
Purity 100 %
Storage conditions At room temperature in the dark
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Females nulliparous and non-pregnant: no
- Age at study initiation: Males: 8 to 10 weeks, Females: 10 to 13 weeks
- Weight at study initiation: Males: 235-348 g, Females: 192-231 g
- Fasting period before study: none
- Housing:
Cages with standard, granulated, S8-15 sawdust bedding (J. Rettenmaier & Söhne)
Premating period (5 animals/cage) Makrolon type IV cages
Mating period (one male and one female/cage) Makrolon type III cages
Postmating, gestation and lactation periods (individual) Makrolon type III cages
- Use of restrainers for preventing ingestion (if dermal): n/a
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Eight days between arrival and pre-treatment start. After acclimatization period, the animals were subjected to a 19-day pretest period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30 and 70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light/12 hours dark.
IN-LIFE DATES: From: 21 February 2018 To: 22 August 2018
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Prepared weekly in Arachis oil and stored at room temperature and in the dark

DIET PREPARATION
- Rate of preparation of diet (frequency): standard Teklad 2014C rat/mouse maintenance diet used
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: not specified

VEHICLE
- Justification for use and choice of vehicle (if other than water): Standard vehicle in these studies
- Concentration in vehicle: n/a
- Amount of vehicle (if gavage): 5mL/Kg
- Lot/batch no. (if required): KMO9422, KMO9047
- Purity: not specified
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: vaginal plug and (or) sperm in vaginal smear
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: not specified
- After successful mating each pregnant female was caged: Females were separated when evidence of mating was detected
- Any other deviations from standard protocol: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The formulations prepared at three different concentrations were analyzed twice over the course of the study to verify that they were correctly prepared. Controls were also analysed to confirm the absence of the test item.
The test item was used as analytical standard.
12-mL aliquots (in duplicate) were taken from each formulation to be analysed.
Duration of treatment / exposure:
5-8 weeks
Frequency of treatment:
Once daily
Details on study schedule:
- F1 parental animals not mated until [...] weeks after selected from the F1 litters: not specified
- Selection of parents from F1 generation when pups were [...] days of age: not specified
- Age at mating of the mated animals in the study: 2 weeks after treatment

Potential indirect exposure to F1 in utero and through the milk during lactation
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (control)
Dose / conc.:
1 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
5 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of GLP Toxicology Study No. 87-XE-ODBP-10 conducted by the U.S. Army Public Health Command and the preliminary results obtained in the previous non-GLP study PX87XC 7-day Oral (Gavage) Dose-Range Toxicity Study for OECD 421 conducted at Envigo CRS, S.A.U.
- The high dose level of 20 mg/kg was chosen with the aim of inducing some developmental and/or maternal toxicity but not death or severe suffering. This dose level was previously tested in toxicology study No. 87-XE-ODBP-10 and produced signs of systemic toxicity but no mortality or suffering. In study PX87XC this dose level showed no evidence of toxicity or mortality.
- The intermediate and low dose levels were selected as a descending sequence to demonstrate any possible exposure-related response.

- Rationale for animal assignment: Random
Positive control:
none
Parental animals: Observations and examinations:
Animals and their cages: Visually inspected twice daily for evidence of reaction to treatment or ill-health.
Blood sampling for hematology or blood chemistry analysis was not performed on animals that died or were sacrificed prematurely.

BODY WEIGHT: Yes
- Time schedule for examinations: Pre-test, Treatment (14 days prior to mating), Mating, Post-mating, Gestation, Lactation

FOOD CONSUMPTION AND COMPOUND INTAKE: weekly

WATER CONSUMPTION AND COMPOUND INTAKE: No
- Time schedule for examinations:

OTHER:
Clinical signs - twice in pre-test, once a week during treatment, mating, and post-mating, day 0, 7, 14 and 20 during gestation, and day 1, 4, 7, and 13 during lactation
Oestrous cyclicity (parental animals):
Dry smears Using inoculation loops during the following phases:
• For 14 days before treatment (all females including spares); animals that fail to exhibit 4-5 day cycles were not be allocated to study.
• Daily from the beginning of treatment period until evidence of mating.
• On the day of necropsy
A cotton swab impregnated in distilled water was used in order to check the evidence of mating during the mating period.
Litter observations:
Clinical observations Observed 24 hours after the considered birth and then daily for evidence of ill-health or reaction to maternal treatment
Litter size Daily from day 1-13 of age
Sex ratio Days 1, 4, 7 and 13 of age
Individual offspring body weights Days 1, 4, 7 and 13 of age
Ano-genital distance Day 1 – all F1 offspring
Nipple/areolae count Day 13 of age – male offspring
Postmortem examinations (parental animals):
F0 Males After final investigations completed (after 5 weeks of treatment)
F0 Females failing to produce viable litter (not pregnant females) Day 25-26 after mating
Females killed at termination Day 14-16 of lactation
Postmortem examinations (offspring):
Selected offspring for thyroid hormone analysis – Day 4 of age (two females per litter where possible)
Scheduled sacrifice - Day 13-15 of age
Statistics:
Bartlett's test, Williams' test, Dunnetts test.
Linear-by-linear test (Cytel, 1995).
Cochran-Armitage test (Cytel, 1995).
Reproductive indices:
Estrous cycle, pre-coital interval, mating performance, fertility and gestation length.
Offspring viability indices:
Survival indexes, litter size, sex ratio, body weight, clinical signs, ano-genital distances or external examination.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
The death of two females (numbers 75 and 66) could be considered occasional and not treatment related given that no mortality was recorded at the high dose, due to the incidence observed in the study and to the fact that no test item related effects were observed in these animals during the study. The major factor contributing to their death remained undetermined histologically.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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:
effects observed, treatment-related
Description (incidence and severity):
T4 and TSH determinations: a decrease was recorded when compared to Control in adult males (at all doses but mainly at 20 mg/kg/day) and females (at 20 mg/kg/day). It was also observed at 20 mg/kg/day in offspring males and females on Day 13 of lactation. Despite the effect observed at 20 mg/kg/day in T4 and TSH determinations, thyroid and parathyroid glands weights were unaffected by treatment. Based on the fact that what was recorded was a decrease in the levels of TSH analyzed and not an increase (which would be related to a possible effect attributable to the components that alter the hormonal function at the level of generation of hypertrophy / follicular hyperplasia) and that no macroscopic findings have been recorded in the pituitary or thyroid / parathyroid glands, this finding cannot be considered as a relevant adverse effect.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
During treatment one female at 1 mg/kg/day (number 62) and one at 5 mg/kg/day (number 76) showed irregular cycles and one female administered at 5 mg/kg/day (number 70) was acyclic.
At termination, all reproductive phase females (pregnant) showed diestrus with the exception of 1/9 (number 68) at 1 mg/kg/day and 2/9 (numbers 70 and 71) at 5 mg/kg/day. These females showed different stages of estrous cycle, which indicated they had recovered the cycle.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
There was no effect of treatment on the pre-coital interval: all animals mated within four days of mating at the first estrous, with the exception of one female at 5 mg/kg/day, whose pre-coital interval extended to 14 days of mating with one male and 2 days of mating with a second male.
Mating performance was 100% for all groups except 5 mg/kg/day males, for which it was 90%.
Fertility was 100% at 0 and 20 mg/kg/day and 90% (9/10) at 1 and 5 mg/kg/day for males and females. These differences cannot be taken into account based on occurrence and lack of dose response.
Gestation length was within the normal range in all treated groups.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No effects seen in any of the parameters tested
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
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:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
There was no effect on offspring growth. There were no offspring clinical or necropsy signs observed which were indicative of a reaction to DNAN, and there was no relevant effect on litter size, sex ratio, survival indices, body weights, ano-genital distance or nipple areolae.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No effects seen in any of the parameters tested
Key result
Reproductive effects observed:
no
Conclusions:
In conclusion, the effects of oral (gavage) administration of DNAN to Wistar rats receiving 1, 5 or 20 mg/kg/day for 14 days prior to mating and until sacrifice can be summarized as follows:
Repeated dose toxicity:
- The No Observed Adverse Effect Level (NOAEL) for repeated dose toxicity was considered to be 20 mg/kg/day for males and females, based on the absence of histopathological findings but the non-considered adverse effects in the T4 and TSH determinations.
Reproductive / developmental toxicity:
- The No Observed Adverse Effect Level (NOAEL) for reproductive / developmental toxicity was considered to be 20 mg/kg/day, taking into account that there was no effect on estrous cycle, pre-coital interval, mating performance, fertility and gestation length or in the offspring on survival indexes, litter size, sex ratio, body weight, clinical signs, ano-genital distances or external examination.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subacute
Species:
rat
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

Developmental toxicity as well as the general systemic toxic potential of DNAN were assessed (2018, Envigo, BC12JY). The study was conducted in accordance with OECD 421 and in compliance with GLP. The animals received doses of DNAN at 1, 5 and 20 mg/kg/day for two weeks prior to mating and up to the day before sacrificing inclusive (males) or up to days 13-15 of lactation (females) which were well tolerated. There were no effects on the offspring on survival indexes, litter size, sex ratio, body weight, clinical signs, ano-genital distances or external examination.

A Study of embryotoxicity and the teratogenicity of 2,4-Dinitroanisole has been performed in rats by oral gavage administration (Gao, 2016, vol 34, pp50-52) following method equivalent or similar to OECD Guideline 414 (Prenatal Developmental Toxicity Study). As the reliability of this study was assessed as Klimish 3 and had major methodological deficiencies, therefore, this study has been reported but can be disregarded.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 November 2017 - 22 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: - OECD 421 Guideline for testing of chemicals adopted 29.07.16: Reproduction/developmental toxicity screening test.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
- Physical form / colour: Pale yellow/solid
- Batch number: 55503625
- Lot number: 02/13
- Purity: 100 %
- Storage conditions: At room temperature in the dark
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Females nulliparous and non-pregnant: no
- Age at study initiation: Males: 8 to 10 weeks, Females: 10 to 13 weeks
- Weight at study initiation: Males: 235-348 g, Females: 192-231 g
- Fasting period before study: none
- Housing:
Cages with standard, granulated, S8-15 sawdust bedding (J. Rettenmaier & Söhne)
Premating period (5 animals/cage) Makrolon type IV cages
Mating period (one male and one female/cage) Makrolon type III cages
Postmating, gestation and lactation periods (individual) Makrolon type III cages
- Use of restrainers for preventing ingestion (if dermal): n/a
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Eight days between arrival and pre-treatment start. After acclimatization period,
the animals were subjected to a 19-day pretest period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30 and 70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light/12 hours dark.
IN-LIFE DATES: From: 21 February 2018 To: 22 August 2018
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Prepared weekly in Arachis oil and stored at room
temperature and in the dark
DIET PREPARATION
- Rate of preparation of diet (frequency): standard Teklad 2014C rat/mouse maintenance diet used
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: not specified
VEHICLE
- Justification for use and choice of vehicle (if other than water): Standard vehicle in these studies
- Concentration in vehicle: n/a
- Amount of vehicle (if gavage): 5mL/Kg
- Lot/batch no. (if required): KMO9422, KMO9047
- Purity: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The formulations prepared at three different concentrations were analyzed twice over the course of
the study to verify that they were correctly prepared. Controls were also analysed to confirm the ab
sence of the test item.
The test item was used as analytical standard.
12-mL aliquots (in duplicate) were taken from each formulation to be analysed.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: vaginal plug and (or) sperm in vaginal smear
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: not specified
- After successful mating each pregnant female was caged: Females were separated when evidence
of mating was detected
- Any other deviations from standard protocol: not specified
Duration of treatment / exposure:
5-8 weeks
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (control)
Dose / conc.:
1 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
5 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of GLP Toxicology Study No. 87-XE-ODBP-10
conducted by the U.S. Army Public Health Command and the preliminary results obtained in the
previous non-GLP study PX87XC 7-day Oral (Gavage) Dose-Range Toxicity Study for OECD 421
conducted at Envigo CRS, S.A.U.
- The high dose level of 20 mg/kg was chosen with the aim of inducing some developmental and/or
maternal toxicity but not death or severe suffering. This dose level was previously tested in toxicology
study No. 87-XE-ODBP-10 and produced signs of systemic toxicity but no mortality or suffering. In
study PX87XC this dose level showed no evidence of toxicity or mortality.
- The intermediate and low dose levels were selected as a descending sequence to demonstrate any
possible exposure-related response.
- Rationale for animal assignment: Random
Statistics:
Bartlett's test, Williams' test, Dunnetts test.
Linear-by-linear test (Cytel, 1995).
Cochran-Armitage test (Cytel, 1995).
Indices:
Reproductive indices: Estrous cycle, pre-coital interval, mating performance, fertility and gestation length.
Offspring viability indices: Survival indexes, litter size, sex ratio, body weight, clinical signs, ano-genital distances or external examination.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
The death of two females (numbers 75 and 66) could be considered occasional and not treatment related given that no mortality was recorded at the high dose, due to the incidence observed in the study and to the fact that no test item related effects were observed in these animals during the study. The major factor contributing to their death remained undetermined histologically.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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:
effects observed, treatment-related
Description (incidence and severity):
T4 and TSH determinations: a decrease was recorded when compared to Control in adult males (at all doses but mainly at 20 mg/kg/day) and females (at 20 mg/kg/day). It was also observed at 20 mg/ kg/day in offspring males and females on Day 13 of lactation. Despite the effect observed at 20 mg/ kg/day in T4 and TSH determinations, thyroid and parathyroid glands weights were unaffected by treatment. Based on the fact that what was recorded was a decrease in the levels of TSH analyzed and not an increase (which would be related to a possible effect attributable to the components that alter the hormonal function at the level of generation of hypertrophy / follicular hyperplasia) and that no macroscopic findings have been recorded in the pituitary or thyroid / parathyroid glands, this finding cannot be considered as a relevant adverse effect.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Body-weight-adjusted prostate, seminal vesicles and coagulating gland and spleen weights in males from Group 4 (20 mg/kg/day), epididymides in males from Groups 3 and 4 (5 and 20 mg/kg/day, respectively) and brain weight in females from Group 4 (20 mg/kg/day) differed significantly when compared to control.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Description (incidence and severity):
During treatment one female at 1 mg/kg/day (number 62) and one at 5 mg/kg/day (number 76) showed irregular cycles and one female administered at 5 mg/kg/day (number 70) was acyclic. At termination, all reproductive phase females (pregnant) showed diestrus with the exception of 1/9
(number 68) at 1 mg/kg/day and 2/9 (numbers 70 and 71) at 5 mg/kg/day. These females showed different stages of estrous cycle, which indicated they had recovered the cycle.
Details on results:
There was no effect of treatment on the pre-coital interval: all animals mated within four days of mating at the first estrous, with the exception of one female at 5 mg/kg/day, whose pre-coital interval extended to 14 days of mating with one male and 2 days of mating with a second male.
Mating performance was 100% for all groups except 5 mg/kg/day males, for which it was 90%. Fertility was 100% at 0 and 20 mg/kg/day and 90% (9/10) at 1 and 5 mg/kg/day for males and females. These differences cannot be taken into account based on occurrence and lack of dose response.
Gestation length was within the normal range in all treated groups.
Number of abortions:
not specified
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
not specified
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not specified
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 20 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
other: No effects seen in any of the parameters tested
Fetal body weight changes:
no effects observed
Changes in sex ratio:
not specified
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
not specified
Skeletal malformations:
not specified
Visceral malformations:
not specified
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No effects seen in any of the parameters tested
Key result
Developmental effects observed:
no

There was no effect on offspring growth. There were no offspring clinical or necropsy signs observed which were indicative of a reaction to DNAN, and there was no relevant effect on litter size, sex ratio, survival indices, body weights, ano-genital distance or nipple areolae.

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for reproductive / developmental toxicity was considered to be 20 mg/kg/day, taking into account that there was no effect on estrous cycle, pre-coital interval, mating performance, fertility and gestation length or in the offspring on survival indexes, litter size, sex ratio, body weight, clinical signs, ano-genital distances or external examination.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
2016
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Appearance: yellow crystalline powder
- Purity: > 99.0%
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Experimental Animal Center of Xi'an Jiaotong University
- Age at study initiation: 13 weeks old
- Diet: nutrient pellets
- Water: ad libitum
- Acclimation period: not stated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3
- Humidity (%): 55 -65
- Photoperiod (hrs dark / hrs light): 12/ 12
Route of administration:
oral: gavage
Vehicle:
other: 4% aqueous starch solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle (if other than water): No justification given, vehicle was 4% aqueous cooked starch solution
- Concentration in vehicle: 0.5, 1.5 and 4.5 mg/mL
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No analytical verification of doses or concentrations reported
Details on mating procedure:
- Number of animal: 120 adult female rats and 60 male rats
- Mating ratio: 1:1
- Proof of pregnancy of pregnancy: vaginal plugs
Duration of treatment / exposure:
Gestation days 5 - 19
Frequency of treatment:
Daily, in the morning
Duration of test:
Rats are sacrified, dislocated on day gestation 20
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
45 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24
Control animals:
yes, concurrent no treatment
yes, historical
Details on study design:
- Rationale for animal assignment: randomly assigned into each of the five groups in the study
Maternal examinations:
OBSERVATIONS: Yes
- Time schedule: daily (the skin, hair color, eyes, mucous membrane, activity)

BODY WEIGHT: Yes / No / No data
- Time schedule for examinations: GD 0, 2, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes / No / No data
- Compound intake calculated as time-weighted averages from the body weight gain data: Yes

POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day: 20
Ovaries and uterine content:
No informationThe ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
Fetal examinations:
No information - External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes
The appearance of the fetal rat is examined. The placenta and body weight of each fetal rat is weighed, their body length, tail length and anal genital nodule spacing are measured.
Statistics:
Statistical analysis is performed using the SPSS 13.0 software. For the measurement data, the t-test is adopted to compare the two groups, and the variance analysis is adopted for comparison between multiple groups. For the classification data, the x2 test is used to compare between groups, and the Dunnet’t test is adopted for the comparison between two groups. The trend of body weight changing with time is analyzed by repeated measurement analysis of variance. If the data does not satisfy the spherical symmetry test, the Greenhouse-Geisser correction will be performed on the degree of freedom. The calibration level is α=0.05
Indices:
No information
Historical control data:
No
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the high-dose group, only, symptoms such as irritability, avoidance, and excitement were observed following dose administration. No effects in the low and medium dose groups.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Compared with the negative control group, there is no significant change in body weight of each dose group, and the difference is not statistically significant (P > 0.05). The net weight increases of pregnant rats at low, medium and high doses are significantly lower than that of the negative control group.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The gross weights of uterus of rats in the high-dose group were lower than those of the negative control group, and the difference was statistically significant (P < 0.05). No treatment related effect in the low or medium dose groups.
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Number of abortions:
not specified
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
The number of dead embryos in the high-dose group is significantly higher than that in the negative control group. The difference is statistically significant (P < 0.05)
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not specified
Other effects:
not specified
Details on maternal toxic effects:
The results of this study indicate that DNAN has maternal toxicity, which is demonstrated by the fact that both the increase in net body weight of pregnant rats and the gross weight of uterus in the low-, medium- and high-dose groups are lower than those in the negative control group.
The gross weights of uterus of rats in the high-dose group are lower than those of the negative control group
Dose descriptor:
LOAEL
Effect level:
ca. 15 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
behaviour (functional findings)
Abnormalities:
effects observed, treatment-related
Localisation:
uterus
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight, body length, tail length and anal genital nodule spacing in the middle- and high-dose groups are all obviously lower than those of the negative control group.
The body weight, body length and tail length of the positive control group were significantly lower than those of the negative control group.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
The number of dead embryos in the high-dose group is significantly higher than that in the negative control group.
The number of live embryos in the positive control group is significantly lower than that in the negative control group.
Changes in sex ratio:
not specified
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
The body weight in the middle- and high-dose groups are statistically (p<0.05) lower than those of the negative control group.
The body weight of the positive control group were significantly lower than those of the negative control group.
Changes in postnatal survival:
not specified
External malformations:
effects observed, treatment-related
Description (incidence and severity):
The body length, tail length and anal genital nodule spacing in the middle- and high-dose groups are all obviously lower than those of the negative control group.
The body weight, body length and tail length of the positive control group are significantly lower than those of the negative control group.

Deformity in appearance of fetal rats in each group:
negative Control: Among the 215 rats, only 1 has appearance deformity with a broken tail.
Low dose group: None of the 205 in low-dose group has deformity in appearance.
Middle dose group: Of the 217 rats in middle dose group, only 1 has deformity in appearance, which is broken tail.
High dose group: None of the 118 rats has deformity in appearance.
Positive control: 26 out of the 193 have deformity in appearance, including 25 with brain swelling, 18 with spina bifida, 4 with short tail, 6 with curly tail, and 2 with abdominal wall fissure.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Negative control: No skeletal deformity is found.
Low dose group: No skeletal deformity is found.
Middle dose group: Of the 120 rats, 6 have skeletal deformity, all of which are rib deformities.
High dose group: Of the 59 rats, 3 have skeletal deformity, including 1 with increased fontanelle size, and 2 with bent ribs.
Positive control: Of the 105 rats, 44 have skeletal deformities, including 37 with increased fontanelle size, 35 with missing occipital bone, 13 with missing 2nd/5th sternum, 11 with fissure in the cervical vertebrae, and 7 with abnormal thoracic vertebrae, 7 with missing ribs and 5 with rib fusion, 2 with abnormal lumbar vertebrae, and 2 with rib bending.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Negative control: Of the 101 rats only 1 has visceral deformity, which is abnormal development of reproductive organs.
Low dose group: 2 of the 103 rats have visceral deformity, all of which are ventricular septal defect.
Middle dose group: Of the 97 rats, 4 have visceral deformity, which are ventriculomegaly, ventricular septal defect, liver abnormality, and gastric calculi.
High dose group: Of the 59 rats, 12 have visceral deformity, including 1 with cleft palate, 7 with ventriculomegaly, 3 with ventricular septal defects, and 2 with abnormal reproductive organs.
Positive control: Of the 88 rats in the positive control group, 42 have visceral deformity, including 38 with ventriculomegaly and 9 with abnormal liver.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
DNAN also has toxic effects on embryos, which is demonstrated by the fact that the number of live fetuses is reduced in the high-dose group, and the number of dead fetuses increases. At the same time, it also has toxic effects on the growth and development of the fetal rats, and such toxic effects increase with the increase of the dose, which is demonstrated by the fact that the body weight, body length, tail length, and anal genital nodule of the middle- and high-dose groups spacing are lower than those of the negative control group, and there is a dose-effect relationship in body weight, body length, tail length and anal genital nodule spacing between groups. In addition, DNAN also has toxic effects on the fetus, which is demonstrated by the increase in the incidence of ventriculomegaly in the high-dose group and the increase in the rib bending rate in the middle-dose group.
Dose descriptor:
LOAEL
Effect level:
ca. 15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations
Abnormalities:
effects observed, treatment-related
Developmental effects observed:
yes
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
Treatment related:
yes
Dose response relationship:
yes

Table 1 Comparison of gestation rate among pregnant rats of each group

Group Number of rats Number of pregnant rats Gestation rate (%)
Negative control group 24 17 70.83
Low-dose group 24 16 66.67
Middle-dose group 24 18 75
High-dose group 24 19 79.17
Positive control group 24 20 83.33
Total 120 90 75

 

Table 2 Body weight, net body weight increase and gross weight of uterus of each group (x±s, g)

Group Number of pregnant rats Body weight of pregnant rats Increase in body weight of pregnant rat Gross weight of uterus
Day 0 Day 2 Day 5 Day 8 Day 11 Day 14 Day 17 Day 20
Negative control group 17 214.53±19.56 229.88±17.84 241.53±18.03 259.82±23.38 271.36±21.36 290.45±21.40 317.58±24.77 365.53±36.71 52.39±18.18 71.61±19.13
Low-dose group 16 216.98±19.81 231.38±21.48 245.79±19.65 257.98±20.51 274.20±18.24 292.14±19.61 324.38±23.15 365.12±29.86 48.45±20.83b 70.88, 20.28
Middle-dose group 18 222.17±22.78 230.72±35.02 248.78±23.03 257.87±23.11 272.93±20.58 288.64±22.01 319.17±23.15 352.65±30.15 38.17±11.90b 65.70±16.76
High-dose group 19 219.73±30.35 223.12±26.83 251.92±26.42 253.61±22.87 267.99±23.71 283.14±25.95 299.36±26.09 297.61±30.67 14.57±24.73b 31.12±15.38b
Positive-control group 20 213.68±21.01 228.39±19.27 245.45±19.80 244.73±19.07 261.66±17.25 276.30±16.85 300.01±20.00 327.85±31.00 37.33±22.26 45.07±20.06b

Compared with the negative control group,aP < 0.05,bP < 0.01

Table 3 Gestation of pregnant rats in each group (x±s)

Group Number of pregnant rats Number of corpus luteums Implantation rate (%) Number of dead embryos Number of absorbed embryos
Negative control group 17 18.53±5.54 74.05±28.60 0.00±0.00 0.35±0.86
Low-dose group 16 17.94±5.67 77.67±28.28 0.00±0.00 0.88±1.54
Middle-dose group 18 19.28±5.00 74.91±20.90 0.50±1.47 0.44±0.86
High-dose group 19 19.95±5.25 70.84±25.01 6.05±5.14b 1.42±2.29
Positive-control group 20 16.15±4.48 80.14±23.49 0.20±0.52 2.70±3.36b

Compared with the negative control group,aP < 0.05,bP < 0.01

Table 4 Growth and development of fetal rats (x ± s)

Group Number of fetal rats Body weight (g) Body length (cm) Tail length (cm) Anal genital nodule spacing (cm)
Negative control group 215 3.79±0.38 3.83±0.18 1.37±0.09 0.20±0.09
Low-dose group 205 3.77±0.38 3.82±0.17 1.38±0.12 0.20±0.09
Middle-dose group 217 3.12±0.50b 3.54±0.24b 1.32±0.09b 0.18±0.07b
High-dose group 118 2.24±0.56b 3.18±0.35b 1.22±0.12b 0.17±0.08b
Positive-control group 193 2.58±0.56b 3.26±0.37b 1.30±0.15b 0.19±0.08

Compared with the negative control group,aP < 0.05,bP < 0.05

Conclusions:
The results of this study indicate that DNAN has maternal reproductive toxicity, embryo toxicity and the teratogenicity to rats however a LOAEL cannot be assigned as there are considerable methodological defects in the performance of the study. It is considered that a guideline study is required to address this endpoint.
Executive summary:

Whilst this study has been performed broadly in accordance to the OECD 414 test guideline there are many points where the data is missing or not considered reliable or fully reported. These include:

No analysis of test solutions, no stability data and no achieved concentration data

No HCD to put the negative or positive control data into context

Individual results are not reported for any endpoint

No calculations of maternal weight gain

No food consumption data

Sex ratio of fetuses and no of live fetuses not recorded

There is doubt over the extent and reliability of the recording of the fetal examinations as all defects are recorded as deformities and there is no recording of any reduced/incomplete ossification which was expected to be present in the positive control group and the medium and high dose groups. Similarly expected visceral markers of immaturity ie. undescended testicles and thyroid, were not reported.

The reliability of the study was therefore assigned as Klimisch 3 and whilst there is evidence that DNAN is a maternal, embryo and fetal toxicant the results cannot be used for classification and a Guideline study is required.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Toxicity to reproduction: other studies

Description of key information

In the subchronic oral toxicity study ( with reliability of Klimish 4)

2.4-dinitroanisole in rats ( US Army Institute of Public Health , 2012, 87-XE-ODBP-10) the treatment caused an apparent increase in metabolism, leading to reduced feed conversion efficiency and reduced body mass gain in males. Anemia, splenic enlargement, hemosiderosis, and extramedullary hematopoeisis (EMH) indicated that the blood is a target organ for DNAN, with females being more sensitive than males. DNAN was found to be a testicular toxicant, causing decreased mass of the testes and epididymides, as well as degeneration and atrophy of the testicular seminiferous tubules and epididymal aspermia. Stereotypical behavior in males, gait irregularities, and cerebellar glial

lesions indicated that DNAN is neurotoxic. A BMDL10 of 2.3mg/kg-day was derived based on EMH in females.

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
September 2010 - March 2011
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Qualifier:
equivalent or similar to
Guideline:
other: OPPTS 870.3100
GLP compliance:
yes (incl. certificate)
Type of method:
in vivo
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
BAE Systems, Ordnance System, 4509 West Stone Drive, Kingsport, TN 37660.
- Expiration date of the lot/batch:
lot BAE10H281-008
- Purity test date:
100%

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
the test material was dried in a vacuum oven at approximately 70°C for 12-48 hours to remove moisture.
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
Dosing solutions/suspensions were prepared by grinding DNAN using a mortar and pestle to a fine consistency, and mixing with a measured volume of cron oil.
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Charles River Laboratories
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation:
8-week old
- Weight at study initiation:
297,1+/-10.88 for males and 214,1+/-9.14g for females
- Fasting period before study:
No
- Housing:
individually housed in suspended polycarbonate cages
- Diet : ad libitum)
- Water : ad libitum
- Acclimation period:
14 days

DETAILS OF FOOD AND WATER QUALITY:
Certified pesticide-free rodent chow and quality drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%):
30-70%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):
12-hour light/dark

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
4 dosing solutions/suspensions were prepared : 0,25, 1, 4 and 16mg/mL. Dosing solutions/suspensions were prepared in volumes sufficient for approximately 2-3 weels of dosing, resulting in preparation of 5 sets of dosing solutions.


- DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):

- Storage temperature of food:

- VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A 1 mL sample was taken from each dosing dolutions/suspensions for analytical verification of concentration of each preparation using a gas chromatograph equipped with an electron capture detector.
Duration of treatment / exposure:
90 days
Frequency of treatment:
All animals were administered at approximately the same time daily, 7 days per week.
Dose / conc.:
1.25 mg/kg bw/day (nominal)
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
80 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 rats/sex/concentration
Control animals:
yes, concurrent vehicle
Details on study design:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
twice daily : once in the morning and one in the afternoon excepts on weekends when observations occured only in the morning
- Cage side observations checked in tables in the attached study report were included.

DETAILED CLINICAL OBSERVATIONS: Ye
- Time schedule:
once in the morning and one in the afternoon excepts on weekends when observations occured only in the morning

BODY WEIGHT: Yes
- Time schedule for examinations:
twice pretest, weekly during the study and the morning of the necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determine: Feedes were reweighted weekly and the mass of the empty feeder was sustracted from the mass of the full feeder to determine the grams of food consumed for each animal
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
pre-study for all animals and animals from the control and the highest dose were examinated within one week of the conclusion of the study.
- Dose groups that were examined:

HAEMATOLOGY/ CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Blood abtained from CO2 anesthetized animals via intracardiac puncture at the termination of the study
- Anaesthetic used for blood collection: Yes CO2
- Animals fasted: Yes overnight prior to bllod collection
- How many animals:
All animals
- Parameters checked in tables in the attached study report were examined.


URINALYSIS: Yes
- Time schedule for collection of urine:
During the last 2 weeks
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, for an overnight period of approximately 12 hours
- Parameters checked in tables in the attached study report were examined

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
at the same time each morning prior to dosing
- Dose groups that were examined:
on each animal prior to initiation of dosing and weekly thereafter with one subset of animals being assessed per day. (animals were divided into 2 subsets for each sex)
- Battery of functions tested: FOB (Functional Observation Battery) and motor activity assessment

IMMUNOLOGY: Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER: SPERM ANALYSIS :
The number of sperm, number of motile sperm, and number of progressive sperm were determined in duplicate for each animal.
Statistics:
Details of the statistical analyses can be found in Appendix T of the study report.
Dose descriptor:
conc. level:
Effect level:
>= 80 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Organ weight findings
In males, testes mass, testes to body mass, and testes to brain mass ratios were reduced in the 80 mg/kg-d group (P<0.001, P<0.001 and P<0.001, respectively).
Epididymides mass and epididymides to brain mass ratio were also reduced, relative to the control, in males given 80 mg/kg-d DNAN (P<0.001 and P<0.001, respectively)

Gross pathology findings:
Small testes were noted in six of the males in the 80 mg/kg-d group. Hydrouterus was noted in five females: one 5 mg/kg-d, two 20 mg/kg-d, and two 80 mg/kg-d

Histopathological findings :
Degeneration and atrophy of testicular seminiferous tubules (moderate to severe) was present in nine of nine males examined in the 80 mg/kg-d group. No test article-related changes were noted in the testes in the control and 20 mg/kg-d groups. Seminiferous tubules of the 80 mg/kg-d group retained only Sertoli cells, spermatogonia and early spermatocytes. Absent germ cell layers included all spermatid and late spermatocyte stages resulting in the absence of mature sperm in seminiferous tubules. Testes additionally demonstrated moderate to numerous numbers of atrophic tubules. Aspermia with eosinophilic cellular tubular debris (moderate to severe) was present in the epididymis of nine of nine males examined in the 80 mg/kg-d group. Few sperm were noted in the cauda of individual animals in the 80 mg/kg-d group.

Sperm Analysis
The number of sperm per gram in the cauda epididymis in male rats in the 80 mg/kg-d group was reduced to 4.5 percent of the number of sperm per gram found in controls (P=0.038). No motile sperm were found in any of the animals in the 80 mg/kg-d group. No significant reductions in sperm per gram, percent motile sperm, or percent progressively motile sperm were observed in the 1.25 , 5, or 20 mg/kg-d dose groups.
Conclusions:
Mortality occurred in three male rats (days 50, 63, and 77) and one female rat (day 26) all from the 80 mg/kg-day dose group.
Decreased mass of the testes and epididymides as well as degeneration and atrophy of the testicular seminiferous tubulesand aspermia were observed in rats from the 80 mg/kg-day group.
As discussed in the repeated dose toxicity study endpoint, this study, the first repetitive oral dosing conducted with DNAN, demonstrated a steep dose response curve, with most effects occurring only in the highest doses and occurring at or near lethal doses.
DNAN demonstrated testicular toxicity that, combined with the documented reproductive/developmental effects of its metabolite, 2,4-DNP, raises concern about the reproductive/developmental toxicity of DNAN.
Executive summary:

In a 90-day oral repeated dose toxicity study, performed according to the OPPTS 870.3100 guidelines and GLP-compliance, 10 rats Sprague-Dawley of each sex were dosed via oral gavage at 0, 1, 25, 5, 20 and 80mg/kg of bw of DNAN, approximately the same time daily, 7 days per week, for 90 days. A vehicule control group of animals was dosed with corn oil at the same volume (5mL/kg) per body mass as the DNAN exposed animals.

Likely owing to its conversion to 2,4 -DNP, an inhibitor of mitochondrial energy homeostasis, DNAN treatment caused an apparent increase in metabolism, leading to reduced feed conversion efficiency and reduced body mass gain in males.

Anemia, splenic enlargement, hemosiderosis and extramedullary hematopoeisis (EMH) indicated that the blood os a target orgna for DNAN, with females being more sensitive than males.

DNAN was a testicular toxicant, causing decreased mass of the testes and epididymides, as well as degeneration and atrophy of the testicular seminiferous tubules and epididymal aspermia.

Stereotypicall behavior in males, gait irregularities, and cerebellar glial lesions indicated that DNAN is neurotoxic.

A BMDL10 of 2,3 mg/kg-day was derived based on the EMH in females.

Justification for classification or non-classification

Substance is not classified toxic to reproduction based on Envigo study, BC12JY, 2018. The supportive study by Gao et al was disregarded as it was Klimisch category 3.