Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1,4-Benzenediamine, N,N’-mixed Ph and tolyl derivs. showed some positive mutagenic findings with bacteria. Its mechanism of mutation induction is through frameshift mutations after metabolisation of the test substance. In vitro in mammalian cells, the test chemical did not induce DNA-damage nor chromosome aberrations. The weak increase in chromosome aberrations noted in the chromosome aberration test is, on basis of the findings noted in a repeat in vitro chromosome aberration test and in an in vivo micronucleus study considered to be not relevant.



Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted per standard procedures for this assay and GLP guidelines
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Published methods including those of Ames (1975) and Green (1976)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Base pair substitution mutations at hisG locus - TA1535 & TA100 salmonella strains; frameshift mutations at the hisD locus - TA1538 & TA98 Salmonella strains; frame shift mutations at hisD locus - TA1537 strain; base pair substitution mutations at trp locus - E. coli WP2 uvrA strain
Test concentrations with justification for top dose:
Ranged from 0.167 to 167 ug/plate. Test article appeared to be partially insoluble >50 ug/plate
Vehicle / solvent:
DMSO was used as it solubilized the test substance to a satisfactory degree, and is commonly used in the Ames assay.
Details on test system and experimental conditions:
Preincubation procedure was used. This entailed mixing aliquot of tester strain, media, and test article - with or without rat liver S9 enzyme system. This combination was incubated at 30 degrees C for 30 minutes. The mixture was then added to molten top agar, and added to minimal glucose plates. Plates were then incubated for 48 hr at 37 degrees C for colony development. Colonies were counted with use of Artek electronic colony counter. Cultures were run in triplicate. Mutations were expressed in the absence of tryptophan (E. coli) or histidine (Salmonella) in incubation media .

A toxicity prescreen was performed in the absence of S9 only in TA 1538, TA100 & E. coli strains. Levels of test material were 50, 167, 500, 1670 & 5000 ug/plate. Test material was not toxic up to level of 167 ug/plate in E. coli. Severe toxicity was seen in Salmonella at 500 ug/plate and at 167 in TA100. Slight to moderate toxicty was seen at 50 ug/plate as judged by reduced background lawn or presence of pindot colonies.
Evaluation criteria:
A positive effect was defined as statistically significant dose-dependent increase in # of revertants with at least one dose level inducing frequency two-fold of spontaneous (solvent) control value. Assay results must be replicated to confirm positive results.
Statistics:
Mean values were calculated for triplicate determinations, and standard deviations calculated. Criteria for positive mutagenic activity requires determination of whether results for test chemical exceed 2x control levels.
Additional information on results:
POTENTIAL CONFOUNDING FACTORS None apparent. It was recognized that test cmpd has low water solubility, and that precipitation was noted & reported in high exposure samples. The range-finding test showed significant cytotoxicity and insolubility at 50 ug/plate or above in 2 of 3 bacterial strains tested . 50 ug/plate was then chosen as high dose for all assays.
Remarks on result:
other: Details on the test results is presented in section "Any other information on results"

Bacterial Mutation Assay 1 ( # revertant)
Dose Level S9 TA1535 TA1537 TA1538 TA98 TA100  E.coli
Solvent  -  5 5 6 21 88 4
0.167  - 6 7 10 22 99  -
0.5  - 5 5 7 19 97  -
1.67  - 7 3 7 14 92  -
5  - 6 6 3 19 90 3
16.7  - 7 a/b 7 a 4 a/b 13 a/b 70 a/b 3
50  - 3 b/c 2 a/b 5 a/b 21 a/b 57 b 4
167  -  -  -  -  -  - 2 a
500  -  -  -  -  -  - 4 a
1000  -  -  -  -  -  - 4 b/c
Solvent  + 5 9 16 26 104 4
0.167  + 7 7 20 30 118  -
0.5  + 5 7 15 27 115  -
1.67  + 7 9 15 22 105  -
5  + 10 6 15 36 99 4
16.7  + 7 11 37 a 47 a 119 a 3
50  + 9 a 14 a/b 94 a 80 a 102 a/b 5
167  +  -  -  -  -  - 3
500  +  -  -  -  -  - 3 a/b
1000  +  -  -  -  -  - 5 a/b
a/b/c - slight/moderate/severe toxicity

Bolded/italics - value 2x control value

Bacterial Mutation Assay - 2 (# revertants

Dose Level

S9 TA1535 TA1537 TA1538 TA98 TA100  E.coli
Solvent  -  8 5 7 23 86 4
0.167  - 11 6 7 22 95  -
0.5  - 8 5 6 24 101  -
1.67  - 5 10 5 17 80  -
5  - 8 5 6 18 86 3
16.7  - 5 a/b 7 a 3 a/b 16 a 48 a 3
50  - 5 b/c 4 a/b 4 a/b 18 a/b 35 b/c 4
167  -  -  -  -  -  - 3
500  -  -  -  -  -  - 3 a
1000  -  -  -  -  -  - 3 a/b
Solvent  + 13 13 13 31 99 3
0.5  + 8 3 18 25 110  -
1.67  + 9 9 16 25 86  -
5  + 13 8 19 23 108 4
16.7  + 9 10 27 32 111 3
50  + 15 9 57 a/b 57 a 103 a 4
167  + 8 a/b 21 a/b 152 a/b 185 a/b 88 a/b 5
500  +  -  -  -  -  - 4
1000  +  -  -  -  -  - 4 a/b
a/b/c - slight/moderate/severe toxicity

Bold italics - 2x control level

Conclusions:
The test material is considered to induce frameshift gene mutations in an Ames (Salmonella) assay in the presence of an enzyme activation system.
Executive summary:

The test substance was subjected to the Ames tests according to standard practices. Through use of positive controls, the bacterial strains used were shown to be active. Increased numbers of revertants were seen in TA1538 & TA98 strains in replicate assays, suggesting mutations were frameshift. This activity was observed only in the presence of S9 activating enzymes. The results for this test substance met criteria for a positive response by virtue of (a) exhibiting dose-responsiveness for # of revertants, (b) one or more concentrations of test chemical induced # of revertants in excess of 2-fold above control values, and (c) results were duplicated in a second assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Consistent with standard practices for conduct of this study, lacks GLP
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
not specified
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Test concentrations with justification for top dose:
Test chemical was added to cell cultures at levels of 0.4, 2.0, 4.0 mg/L without S9 enzymes, and 2.5, 12.5 & 25 mg/L with S9.
Vehicle / solvent:
Acetone was used as test chemical vehicle. All culture flasks contained level of 0.5% acetone following test chemical addition..
Details on test system and experimental conditions:
Test system employed cell line derived from Chinese hamster ovaries designated CHO-K1-BH4, lot #M7. Cells were obtained from Oak Ridge Laboratories, Tennessee. S9 hepatic enzymes were derived from male rats, and supplemented with cofactors needed for oxidative metabolism.. Cytotoxicity trials were conducted by exposing plated cells in cultures in a rangefinding test up to level of 375 mg/L, well above the solubility limit of the test chemical. Positive/negative controls were included in the trials as was +/- S9 activation. Exposures were 5 hrs which was followed by rinsing of cells, then addition of BrdU for incubation for an additional 28.5 hrs.

A second trial was performed as above except no S9 was added, and chemical exposures were 28.5 hrs as were exposures to BrdU. A third trial was conducted with chemical exposures to cell cultures in the absence of BrdU for 5 hrs (+/- S9), and for 24 hrs (- S9).

Colcemid was added to each culture for last 2-3 hrs of incubation to arrest cells in metaphase. These cells were collected either by mitotic "shake-off" method or by collecting the entire population of cells. Metaphase plates were prepared on glass slides & stained with fluorescence-plus Giemsa, allowing identification of each metaphase by differential staining of chromosomes. 1000 cells per culture are scored for first, second, third metaphase divisions, and from these values, "average proliferation times" and mitotic indices were calculated.

To assess chromosomal aberrations, CHO cells in logarithmic phase of growth are plated in culture chambers at density of ~800,000 cells per cm2. In "Schedule 1" experiment, chemical, controls, & S9 exposures were 5 hrs followed by 18 hr incubation prior to harvesting of cells. In Schedule 2 & 3, cells were exposed to all chemicals but not S9, for 24 and 48 hrs, respectively. As in cytotoxicity phase, all sets were exposed to colcimid during last 2-3 hrs of incubation. Cells were then collected with shake-off technique, centrifuged, resuspended in hypotonic KCl, and incubated at 37C to allow swellling of cells. Cells were then fixed, and metaphase spreads prepared on glass slides. Each was stained in Giemsa solution followed by mounting.

200 well-spread metaphases were pooled and scored for presence of chromosomal aberrations in bright field microscope. Cells were located by scanning slide under low power. Those judged acceptable for analhysis based on morphology & total centromere # (20 +/- 2) were further analyzed with 100x magnification. Abnormalities were classified according to chromosome- or chromatid-type aberrations. Gaps were not included in statistical analyses.

The study was done in duplicate.
Evaluation criteria:
A positive action on the part of test chemical is based upon its ability to induce statistically significant dose-related increase in chromosomal aberrations in at least the two highest doses and/or in the proportion of aberrant metaphases as compared to solvent control. A response not producing either statistically significant dose-related increase in number of structural chromosomal aberrations or a significant and reproduciblepositive response at any test points is considered a negative (non-clastogenic) outcome. Other cirteria include (a) solvent control must have <0.09 aberrations per cell, and (b) positive controls must show statistically significant increases in aberration frequency and in proportion of aberrant metaphases as compared to solvent control.
Statistics:
Chi-square analyses are done to compare proportions of cells with one or more aberrations relative to solvent controls. One-tailed t-tests are used to assess differences in aberrations/cell for treated groups versus solvent controls.
Additional information on results:
Because pH & osmolality are known to impact this assay, these parameters were measured before & after incubations. The pH range at initial time point was 8.41 - 8.51, and at post-treatment, 7.11 - 7.28. Mean initial osmolality (mOSM/L water) ranged from 285 - 368.
Remarks on result:
other: Details on test results are provided in section "Any other information on results"

DAPD - Induction of Cellular Aberrations

Compound Dose - ug/mL S9 Treat (hrs) Aberrations/cell t-Test
Untreated 0  - 5 0.025 (0.157) NS
Acetone 0.50%  - 5 0.015 (0.122)  -
DAPD 0.4  - 5 0.015 (0.122) NS
" 2  - 5 0.040 (0.196) NS
" 4  - 5 0.005 (0.071) NS
MNNG 1.5  - 5 0.500 (0.802) p<0.01
Untreated 0  + 5 0.035 (0.253) NS
Acetone 0.50%  + 5 0.020 (0.140)  -
DAPD 2.5  + 5 0.020 (0.140) NS
" 12.5  + 5 0.020 (0.140) NS
" 25  + 5 0.060 ((0.238) p<0.05
DMN 1000  + 5 0.975 (1.0389) p<0.01
Untreated  -  - 24 0.010 (0.100) NS
Acetone 0.50%  - 24 0.010 (0.100)  -
DAPD 0.4  - 24 0.025 (0.157) NS
" 2  - 24 0.050 (0.218) p<0.05
" 4  - 24 0.070 (0.275) p<0.01
Untreated  -  - 48 0.025 (90.157) NS
Acetone 0.50%  - 48 0.060 (0.238)  -
DAPD 0.4  - 48 0.015 (0.122) NS
" 2  - 48 0.015 (0.122) NS
" 4  - 48 0.060 (0.238) NS

NS - "Not significant"

DAPD - Induction of Chrosomal Aberrations (200 Metaphases) Confirmatory Assay
Compound Dose - ug/mL S9 Treat (hrs) Chromatid Ab./ Exchange Chromosome Ab. Total Aberrations* Chromatid Ab./ Exchange Chromosome Ab. Total Aberrations*
Untreated 0  - 5 6 0 5 Not repeated
Acetone 0.50%  - 5 3 1 3
DAPD 0.4  - 5 3 0 3
" 2  - 5 2 4 8
" 4  - 5 1 1 1
MNNG 1.5  - 5 75 25 100
Untreated 0  + 5 10 0 7 6 5 8
Acetone 0.50%  + 5 4 1 4 13 1 7
DAPD 2.5  + 5 6 0 4 - - -
" 12.5  + 5 5 0 4 - - -
" 25  + 5 11 4 12 13 0 11
DMN 1000  + 5 164 35 195 124 32 150
Untreated  -  - 24 6 0 2 6 0 5
Acetone 0.50%  - 24 2 0 2 4 1 3
DAPD 0.4  - 24 5 0 5 2 1 3
" 2  - 24 9 3 10 6 3 9
" 4  - 24 12 4 14 5 1 2
Untreated  -  - 48 6 1 5 Not repeated
Acetone 0.50%  - 48 14 2 12
DAPD 0.4  - 48 3 0 3
" 2  - 48 3 1 3
" 4  - 48 10 1 12

* Totals do not include gaps. Not considered true chromosomal aberrations.

Conclusions:
DAPD did not induce any chromosomal aberrations in in vitro CHO cells.
Executive summary:

DAPD was subjected to incubations with mammalian cells, a cell line from Chinese hamster ovaries, in the presence of hepatic S9 activation enzymes. Following establishment of a dose range that included a cytotoxic impact as measured by the mitotic index, chemical exposures were chosen to assess the chemical's potential to affect chromosomal structure in these cells. Following various exposure conditions under which positive control substances responded, DAPD was shown to lack any clastogenic activity in this assay system.

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted per GLP & OECD test guidelines
Qualifier:
according to
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Target gene:
Not applicable
Test concentrations with justification for top dose:
Material dose levels were considered acceptable based upon appearance of turbidity, other signs of insolubility. Replicate assays were performed. Exposures ranges in 2 studies: DAPD - 2.0 & 0.4 mg/L, and 2.0, 4.0, 8.0, 16 mg/L; R-59 - 0.4 & 2.0 mg/L, and 2.0, 4.0, 8.0, 16 mg/L; R-898 - 0.04, 0.2, 1.0, 5.0 mg/L and 0.5, 1.0, 5.0, 8.0, 16 mg/L.
Vehicle / solvent:
DMSO was the solvent for all materials, and was in test media at level of 1%.
Details on test system and experimental conditions:
Hepatocytes were isolated from 2 adult Fischer 344 rats (180-200 g) from Charles River Labs, North Carolina. Following anesthetization with Nembutal sodium, a canula was inserted into hepatic portal vein, and the liver perfused with pH 7.4 perfusion media suitable for cell collections (containing collagenase). Cells were dispersed into bovine serum albumin solution, filtered through gauze, and centrifuged. After resuspensions & centrifugations, cells were checked for viability using Trypan Blue stain. One ml aliquots of 500,000 cells per ml were dispensied into culture dishes containing a collagen-coated coverslip & 2 ml of William's media. Cells were allowed to attach for 2 hr in 95% air, 5% CO2 incubator at 37 degrees C.

Two hrs later, cells were rinsed with serum-free Williams media, and test chemicals & 3H-thymidine were added to each culture well for period up to 20 hrs. Cells were then rinsed, fixed, and then exposed to autoradiographic emulsion. The slides were developed after 7 days, and then stained with H&E.

Cytotoxicity was assessed by counting cells remaining attached to the glass coverslip using an Artek Counter. Radiographic grains were counted in 50 randomly-chosen nuclei per slide, 3 slides per chemical concentration. Cells that were undergoing replicative synthesis, or toxicity (irregularly shaped nuclei or nuclei without surrounding cytoplasm) were not counted. Net nuclear grain counts (NNG) were calculated by subtracting the average of 3 cytoplasmic grain counts from the nuclear grain counts. The average NNG counts from 3 slides for each chemical/concentration, and a standard deviation calculated. When results were negative for 2 concentrations showing no toxicity, lower does were not counted.

The study was duplicated for all test materials.

Evaluation criteria:
Assay is valide when solvent/negative controls yield negative values (<0) while positive controls yield positive results (>0). In addition, the positive control values must be statistifcally greater than those of negative controls. In addition, at least 2 concentration yield NNG counts significantly greater than concurrent negative controls, there should be positive dose-responsiveness for increases of NNG up to toxic levels, and at least one of the increased NNG counts is = to, or > than 5. Results for test substance is reported negative if all criteria are not met. It is equivocal if NNG counts are between 0 & 5.
Statistics:
NNG counts for test substance versus negative controls were analyzed using Student's t-test.
Remarks on result:
other: Details on test results are presented in section "Any other information on results"

UDS RESULTS FOR DAPD, R-59 & R-898 (Test #2)
Chemical Conc. (mg/L) NNGs Evaluation
Neg controls
Cells*  - -24.4 Negative
DMSO 1% -37.8 Negative
Fluorene 10 uM -45.2 Negative
Positive control
 2-AF** 10 uM 59.0 Positive
DAPD 0.10 -40.3 Negative
0.25 -30.4 Negative
0.50 -30.6 Negative
1.0 -32 Negative
2.0 -26.1 Negative
4.0 -19.2 Toxic
8.0 -21.4 Toxic
16.0 -4.6 Toxic
R-59 0.50 -36.7 Negative
1.0 -34.1 Negative
2.0 -16.4 Negative
4.0 -22.5 Toxic
8.0 -11.9 Toxic
16.0  - Toxic
R-898
0.25 -31.2 Negative
0.50 -35.6 Negative
1.0 -24.3 Toxic
5.0 -26.8 Toxic
8.0 -17.1 Toxic
16.0 -15 Toxic

* Cells, no solvent ** 2 -Aminofluorene

Conclusions:
The submission substance (DAPD) and 2 of its major components, R-59 & R-898, subjected to an unscheduled DNA repair synthesis assay, displayed an absence of DNA-damaging potential by any of the 3 substances tested.
Executive summary:

The submission substance (DAPD) and 2 of its major components, R-59 & R-898, were subjected to an unscheduled DNA repair synthesis assay using primary hepatocytes from rats. The study was conducted according to OECD test & GLP guidelines. This testing was done in duplicate, and chemical levels in the assay were employed that exceeded solubility limits and/or attained toxic levels. The results indicate an absence of DNA-damaging potential by either the multi constituent substance, or either of 2 of the major components of this substance.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

An in vivo micronucleus test in CD-1 mice was clearly negative when 1,4-benzenediamine, N,N’-mixed Ph and tolyl derivs. was tested up to lethal dose levels.

An in vivo Comet assay in male Sprague-Dawley rats did not show any evidence of causing an increase in DNA strand breaks in the liver and the urinary bladder up to oral dose levels of 1000 mg/kg/day. In the glandular stomach at the dose level of 1000 mg/kg/day, an increase in DNA strand breaks was noted. However, histopathology findings would suggest that the increases in DNA damage observed may be as a result of direct irritation on the glandular stomach. Therefore, the DNA strand breaks observed in the glandular stomach are inconclusive for a true genotoxic response.


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Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study; study run comparable to regulatory guidelines.
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
Mice were obtained from Charles River Labs, Massachusetts. Mice were held in quarantine for 25 days prior to study initiation at which time, the mice were approximately 10 wks of age. Males weighed 27-44 g, and females 27-35 g. Mice were randomly assigned to one of five study groups, 5 males & 5 females per group. Holding rooms were maintained at 22 +/-degrees C, and 12 hr light/12 hr dark cycle. Relative humidity was 55 +/- 15%. Water & diet (Harlan Teklad Rodent Diet) was provided ad libitum. A maximum of 5 mice were housed in wire mesh cages throughout study.

The in-life phase was conducted from 1993-05-11 to 1993-06-17.
Route of administration:
intraperitoneal
Vehicle:
The dosing solvent was mixture of DMSO & corn oil, approximately 1:6 ratio. This produced dosing suspensions of DAPD with concentrations of test material of 25, 125, & 250 mg/10 ml. Delivered dosing volumes was 10 ml/kg.
Details on exposure:
A single intraperitoneal dose was administered at a volume of 10 ml/kg BW. Delivered doses were 250, 1250 & 2500 mg/kg for test animals. Negative controls received solvent only
Duration of treatment / exposure:
Single exposures administered.
Frequency of treatment:
One dose of control and test substance was administered.
Post exposure period:
At each dose level, mice were grouped to provide 5 males, 5 females for sampling at 3 time periods - 24, 48, & 72 hrs post dosing.
No. of animals per sex per dose:
Five mice per sex per dose.
Positive control(s):
Triethylenemelamine (TEM) was administered at dose of 0.5 mg/kg in saline solution.
Tissues and cell types examined:
Bone marrow:
Erythropoietic cells were removed from the femur marrow canal of mice at sacrifice. Cells with micronuclei were graded as polychromatic MPCEs) or normochromatic (MNCEs) erythrocytes.
Details of tissue and slide preparation:
At sacrifice, marrow cells were removed from femurs by perfusing bone channel with fetal bovine serum. Collected cells were concentrated with centrifugation followed by splacing a drop of the cell suspension onto a glass slide. Smears were dried, dipped in methanol, and air-dried. Staining was performed with a Modified Wrights Stain Pak containing polychrome methylene blue-eosin. Scoring of slides were performed in a blind design.

Cytogenetic analysis: an erythrocyte containing one or more micronuclei was consider a micronucleated erythrocyte. 1000 PCEs per mouse were scored for presence of MPCEs as well as # of MNCEs contained within the 1000-PCE field. Data were then expressed as number & % of MPCEs for 10,000 PCEs per group.
Evaluation criteria:
An acceptable study resulted when (a) frequency of MPCEs for negative control is <0.25%, (b) this value for positive control group is statistically increased (p<0.05) compared to negative control values, and (c) a minimum of 7 mice per dose level survive to be scored. A depression in the PCE/NCE ratio indicates a toxic response by a test chemical.
Statistics:
A positive response is when there is increase in incidence of MPCEs in the test chemical group(s) compared to negative controls as determined by a one-tailed t-test.
Additional information on results:
A rangefinding study was conducted under same conditions as described for definitive study. Dose levels were administered to 4 mice per group at doses from 250 to 5000 mg/kg BW. One of 4 mice died at the 5000 mg/kg dose. Mice also exhibited writhing, piloerection, ptosis & decreased activity at this dose level over a 72 hr observation period. Based upon this finding, doses used in definitive study were 250, 1250 & 2500 mg/kg.

In the definitive study, 2 deaths were observed in the 1250 mg/kg group, and 3 deaths in the 2500 mg/kg group. Piloerection, abnormal stance, and writhing were noted in all dose groups, but in a dose-related trend. Results of the erythrocytes evaluations for micronuclei are presented in table below.

DAPD - Induction of Micronuclei in Erythrocytes in vivo
Compound Dose - mg/kg Sac. Time (hr) # Mice MPCEs-%(S.D.)* t-Test PCE/NCE Ratio
Solvent 10 mL/kg 24 10 0.040 (0.052)  - 0.972
DAPD 250 24 10 0.050 (0.053) NS 0.977
" 1250 24 10 0.060 (0.052) NS 0.948
" 2500 24 10 0.060 (0.070) NS 1.024
TEM 0.5 24 10 2.540 (1.213) p<0.01 1.035
Solvent 10 mL/kg 48 10 0.040 (0.052)  - 1.293
DAPD 250 48 10 0.050 (0.070) NS 1.323
" 1250 48 10 0.075 (0.046) NS 1.141
" 2500 48 10 0.050 (0.071) NS 0.906 (p<0.01)
Solvent 10 mL/kg 72 10 0.090 (0.074)  - 0.91
DAPD 250 72 10 0.060 (0.070) NS 0.97
" 1250 72 10 0.040 (0.052) NS 0.843
" 2500 72 10 0.057 (0.079) NS 1.256

* Micronucleated polychromatic erythrocytes - percentage compared to total

polychromatic erythrocytes

Conclusions:
Interpretation of results (migrated information): negative
DAPD was not found to possess clastogenic activity in this mouse erythropoietic cell in vivo assay for micronuclei.
Executive summary:

DAPD was administered to mice by intraperitoneal injections at doses that included toxic responses (lethality, behavioural effects, and decreased PCE/NCE ratio). Erythrocytes were isolated from the femur marrow at periods up to 72 hr post dosing. These cells were assessed for evidence of micronuclei induction in PCEs with an increase seen only for the positive control TEM. DAPD was not found to possess clastogenic activity in this mouse erythropoietic cell in vivo assay for micronuclei.

Endpoint:
genetic toxicity in vivo, other
Remarks:
In Vivo Comet Assay
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017 / 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study results partly inconclusive
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Qualifier:
according to
Guideline:
other: ICH (2011) EMA/CHMP/ICH/126642/2008. Guideline S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use
Qualifier:
according to
Guideline:
other: EC Commission Regulation No. 2017/735. Method B.62: Mutagenicity – In Vivo Mammalian Alkaline Comet Assay. OJ L 112/180.
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Niagara Falls Chemical Plant 5500 Goodyear Drive Niagara Falls, New York. Batch number: 14304 501601343001
- Expiration date of the lot/batch: Test every 36 months via chemical Assay.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature 15 - 25°C

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

OTHER SPECIFICS: Stability and homogeneity of the DAPD in the vehicle were not determined in this test and remain the responsibility of the Sponsor. Chemical analysis of dosing formulations for achieved concentration was not performed in this study.
Alternate name: POLYSTAY 100.
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England.
- Age at study initiation:
Preliminary toxicity test:
On Dispatch:Males and females ca 42 - 49 days old
Day 1:Males and females ca 47 - 55 days old.
Comet test:
On Dispatch: Males ca 42 - 49 days old.
Day 1: Males ca 47 - 54 days old.
Weight at study initiation:
Preliminary toxicity test:
Males weighed between 194 g to 213 g.
Females weighed between 176 g to 194 g.
Comet test:
Males weighed between 177 g to 211 g.
- Assigned to test groups randomly: yes
- Diet (e.g. ad libitum): pelleted Envigo Teklad 2014C ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 40 to 70%
- Photoperiod (hrs dark / hrs light): The room was illuminated by artificial light for 12 hours per day.

EXPERIMENTAL PERIOD: From: 20 July 2017To: 8 Febaruary 2018
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil Westmill Foods
- Justification for choice of solvent/vehicle: solubility of test item
- Lot/batch no. L2 6005/AF/10:59
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The preliminary toxicity test was performed to identify the maximum tolerated dose (MTD), defined as the highest dose that will be tolerated without evidence of study-limiting toxicity, relative to the duration of the study period (for example, by inducing body weight depression or hematopoietic system cytotoxicity, but not death or evidence of pain, suffering or distress necessitating humane euthanasia) up to a standard limit of 2000 mg/kg/day.
The experimental design is shown below:
Group Treatment Concentration Dosage Number of animals
(mg/mL) (mg/kg/day) Males Females
1a DAPD 200 2000 2 2
1 DAPD 100 2000 2 2
2 DAPD 50 1000 2 2
a-Animals killed on Day 2; it was considered they did not receive the full dosage on Day 1 and could not be dosed on Day 2 due to the consistency of the formulation.
Following dosing, the animals were examined regularly during the working day; any mortalities or clinical signs of reaction during the experiment were recorded. At the end of this observation period, surviving animals were killed and discarded.
During the first preliminary toxicity test it was noticed prior to dosing the initial group 1 animals on Day 2 that a large amount of semi-solid material was caught in the rubber catheter and therefore could not be dosed. It also could not be confirmed that the animals on Day 1 received the full dosage. Therefore the formulation method and dose volume was amended in order to ensure a homogenous suspension was achieved. The data generated from these animals will not be reported.

Duration of treatment / exposure:
24 hours
Frequency of treatment:
two occasions approximately
Post exposure period:
3h after second dose
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Group 2 = 12.5 mg/mL
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Group 3= 25 mg/mL
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4 = 50mg/mL
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
ethylmethanesulphonate
- Route of administration: oral gavage, dosed oncy only approximately 4 hours prior to termination
- Doses / concentrations: dose volume of 10 mL/kg (200mg/kg/day)
Tissues and cell types examined:
Kidneys, liver, glandular stomach and urinary bladder.
Details of tissue and slide preparation:
The kidneys were examined macroscopically for adverse findings and stored in 10% buffered formalin.Sections of the liver, glandular stomach and urinary bladder were placed into ice cold mincing solution; all samples were stored on ice before processing for Comet analysis. Single cell suspensions were prepared using a tissue specific method detailed below:
Urinary bladder-The urinary bladder was removed and placed into a Petri dish. It was cut in half and washed in 2 mL of fresh complete mincing solution. An additional 1 mL of complete mincing solution was added and the urinary bladder cut into several small pieces and poured into a falcon tube. The sample was stored on ice until slide preparation.
Liver-Approximately 0.5 cm3 of liver was cut into several small pieces and washed in fresh complete mincing solution until as much blood was removed as possible. The pieces were transferred to 150 µm bolting cloth, 2 mL of fresh complete mincing solution was added and the liver was pushed through the cloth. An additional 2 mL of complete mincing solution was added and any remaining liver pushed through the cloth. The sample was stored on ice until slide preparation.
Glandula stomach-The stomach was removed, cut open and washed free of food using complete mincing solution. The glandular stomach was placed into a falcon tube and covered with approximately 2 mL of mincing solution. The samples were incubated on ice for approximately 30 minutes (± 10 minutes). After incubation the tissue was removed and the surface epithelia was gently scraped using a cell scraper; this layer was discarded and the stomach rinsed. The tissue was transferred to a clean Petri dish, 2 mL of complete mincing solution was added and the stomach was carefully scraped 4-5 times using a cell scraper to release the cells. The cells were poured into a clean falcon tube and stored on ice until slide preparation.

DETAILS OF SLIDE PREPARATION:
Glass slides were dipped in 1% normal melting point agarose and left to air dry prior to addition of the cell suspension layer.
For each tissue type, an appropriate dilution of the cell suspensions were made and mixed with the appropriate volume of 0.5% low melting point agarose. A 75µL aliquot of the cell/agar mix was dispensed onto the appropriate pre-dipped slides and cover-slipped.
Once the agar had set the cover slips were removed and the slides immersed in chilled lysis solution in a light proof box. These were stored at 2 - 8ºC overnight prior to electrophoresis.

METHOD OF ANALYSIS:
Electrophoresis-The slides were rinsed in electrophoresis buffer and randomly placed onto a dry, level platform of a horizontal electrophoresis unit containing chilled electrophoresis buffer. The slides from each treatment were spread across the platform to avoid any positional effects.The buffer reservoir of the unit was topped up with electrophoresis buffer until the surfaces of the slides are covered. The slides were left for 20 minutes to allow the DNA to unwind. After alkali unwinding, the slides were electrophoresed at 18V with a starting current of approx. 300 mA (between 0.7 to 1.0V/cm) for 30 minutes.The temperature of the electrophoresis solution during unwinding and electrophoresis was maintained at a low temperature (4 – 8°C).When electrophoresis was completed, the slides were washed 3 times, for 5 minutes with neutralisation buffer and stored, refrigerated, in lightproof boxes with moistened tissues (to prevent agar dehydration).
Microscopic Examination- Coded slides were examined by staining with SYBR GOLD® and visualised under a fluorescence microscope. The comet images from the microscope were captured via a CCD camera and measured using Perceptive Instruments COMET IVTM image analysis system.Initially the slides were examined for any overt toxicity, e.g. an increase in background debris and/or an increase in the incidence of excessively damaged cells (i.e. ‘hedgehog’ or ‘ghost’ cells). These cells were excluded from the analysis, along with any cells that had unusual staining artefacts. Fifty cells were scored per slide to give a total number of 150 cells per tissue per animal. The extent of DNA migration and hence damage is reflected by: % TAIL INTENSITY, defined as the fluorescence detected by image analysis in the tail, which is proportional to the amount of DNA that has moved from the head region into the comet tail.
Pathology- Histopathological examination was performed on sections of the glandular stomach from the vehicle control and animals administered DAPD at 250, 500 and 1000 mg/kg/day (fixed in 10% buffered formalin). Samples were processed to paraffin wax, sectioned at approximately 4 5 µm and stained with haematoxylin and eosin. Processed samples were examined by light microscopy for evidence of cytotoxicity.Sections of the liver, urinary bladder, kidney and glandular stomach samples which were not processed by pathology were stored in 10% buffered formalin and stored within Cell and Molecular Sciences.
Major Computerized Systems - Pharmacy TEST ITEM management: Pristima.Statistics: Quasar.Slide analysis: COMET IVTM Image analysis system.




Evaluation criteria:
The following criteria were applied for assessment of assay acceptability:
The concurrent vehicle control is considered acceptable for addition to the laboratory historical vehicle control database.
Concurrent positive control should induce responses that are compatible with those generated in the historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
A maximum tolerated dose or maximum feasible dose has been achieved.
Adequate numbers of cells and doses have been analysed.
Statistics:
Separate analyses were performed for Groups 1, 2, 3 and 4 and Groups 1 and 5.
For Groups 1, 2, 3 and 4, Bartlett’s test for variance homogeneitywas applied. If this test was not significant at the 1% level, then parametric analysis was applied. The F1 approximate test was applied to Groups 1 to 4. If the F1 test was not significant at the 1% level, Williams' test for a monotonic trend was applied to compareGroups 2, 3 and 4 to the vehicle control group; otherwise Dunnett's test (Dunnett 1955, 1964) was performed instead. If Bartlett's test was significant at the 1% level, then logarithmic and square-root transformations were tried. If Bartlett's test was still significant, then non-parametric tests were applied. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied to Groups 1 to 4. If the H1 test was not significant at the 1% level, Shirley's test for a monotonic trend (Shirley 1977) was applied to compare Groups 2, 3 and 4 to the vehicle control group, otherwise Steel's test (Steel 1959) was performed instead.
See continued explantation below.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: liver
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: urinary bladder
Sex:
male
Genotoxicity:
ambiguous
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: glandular stomach cytotoxicity, irritation and inflammation
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000-2000 mg/kg bw
- Solubility: Suspensions of DAPD were prepared in corn oil (100 and 50 mg/mL for the 2000 and 1000 mg/kg bw groups)
Note: During a first preliminary toxicity test it was noticed prior to dosing the initial group 1 animals on Day 2 (for a 200 m/mL sustpension) 2 that a large amount of semi-solid material was caught in the rubber catheter and therefore could not be dosed. It also could not be confirmed that the animals on Day 1 received the full dosage. Therefore the formulation method and dose volume was amended in order to ensure a homogenous suspension was achieved. The data generated from these animals were not reported.
- Clinical signs of toxicity in test animals:
To determine suitable dose levels for use in the comet test, one group consisting of two male and two female animals were administered DAPD at 2000 mg/kg/day on two occasions approximately 24 hours apart. At 2000 mg/kg/day clinical signs of toxicity observed in male animals included flattened and hunched posture, piloerection, unsteady gait, decreased activity, unresponsive, abnormally cold to touch and red discharge from left eye. Both male animals were killed post-dose on Day 1 due to the adverse clinical signs observed. At 2000 mg/kg/day clinical signs of toxicity observed in female animals included reduced body tone, unsteady and elevated gait, hunched posture, piloerection and lachrymation. Both female animals were killed post-dose on Day 1 due to the adverse clinical signs observed. On the basis of this result the maximum tolerated dose had been exceeded in both male and female animals; therefore an additional group of two male and two female animals were administered DAPD at 1000 mg/kg/day. At 1000 mg/kg/day clinical signs of toxicity observed in male animals included hunched posture, elevated gait, piloerection, loose feces, dark coloured feces and fecal staining. Both male animals survived until scheduled termination. At 1000 mg/kg/day clinical signs of toxicity observed in female animals included hunched posture, elevated gait, piloerection, abnormally cold to touch, loose feces, dark coloured feces and fecal staining. DAPD administered at 1000 mg/kg was therefore considered to be the maximum tolerated dose in both male and female animals. No substantial differences in toxicity were observed between the sexes, therefore, in line with current guidelines the comet test was performed using male animals only, dose levels of 250, 500 and 1000 mg/kg/day were selected. Some incidences of body weight loss were observed throughout the preliminary toxicity testing.
- Rationale for exposure: clinical observations indicated that the substance was systemically available.

RESULTS OF DEFINITIVE STUDY
The comet test was carried out in male animals only. No mortalities were observed throughout the duration of the comet test.Table 3 gives a summary of the results of the comet test and the results of statistical analysis
2.1- Clinical Signs
Animals were treated with DAPD at dose levels of 250, 500 and 1000 mg/kg/day. No clinical signs of toxicity were observed for the vehicle or positive control animals over the duration of the test. At 250 mg/kg/day clinical signs of toxicity observed included hunched posture, unsteady and elevated gait, fecal staining and brown anal staining. At 500 mg//kg/day clinical signs of toxicity observed included hunched posture, elevated gait, piloerection, abnormally cold to touch, decreased activity and brown anal staining.At 1000 mg//kg/day clinical signs of toxicity observed included hunched posture, elevated and unsteady gait, piloerection, decreased activity, abnormally cold to touch and brown anal staining. Some incidences of body weight loss were observed in all groups throughout the comet test.
2.2 - Macroscopic kidney examination
The kidneys were examined macroscopically for adverse findings. Pale kidneys were observed in two animals administered DAPD at 500 mg/kg/day and in one animal administered DAPD at 1000 mg/kg/day. No other abnormalities in the kidneys were detected.
2.3 -% Tail Intensity - See Tables 1 (Liver), 2 (Glandular stomach), 3 (Urinary bladder) below.
There were no statistically significant increases in the median % tail intensity (%TI) observed in the liver or urinary bladder of male Crl:CD(SD) rats when administered DAPD at any dose level compared to vehicle control values. The liver group mean and median %TI of all treatment groups were within the current historical control range. The urinary bladder mean % TI of all treatment groups did not exceed the current historical control range.Statistically significant increases in the median % TI were observed in the glandular stomach of male Crl:CD(SD) rats when administered DAPD at 500 and 1000 mg/kg/day (p<0.001) compared to vehicle control values. The group mean % TI value of animals administered DAPD at 1000 mg/kg/day is outside of the current vehicle historical control range. No changes were observed at 250 mg/kg/day. The positive control compound, Ethyl methanesulfonate, produced statistically significant increases in the median % TI when compared to vehicle control values (p<0.001, t-test).
2.4 - Hedgehog “Ghost” Cell Data
No hedgehog cells were observed in male Crl:CD(SD) rats administered DAPD at any dose level, in all tissues analysed.
2.5 - Pathology
Microscopic findings related to treatment with DAPD were observed in the glandular stomach of male rats given doses of 500 or 1000 mg/kg/day, consisting of minimal to slight focal/multifocal necrosis or erosion of the mucosa, minimal inflammatory cell infiltration of the mucosa and/or submucosa, and minimal to slight submucosal oedema.Additionally, histopathological findings considered related to treatment were seen in co-processed non-glandular stomach (one animal given 250 mg/kg/day and two animals given 500 mg/kg/day) and pylorus (one animal given 1000 mg/kg/day).The spectrum of histopathological findings in the stomach were considered consistent with direct irritation by the test item DAPD, delivered by oral gavage





COMET ASSAY

Table 1. Comets - Liver

Treatment

Dose
(mg/kg/day)

Number of cells scored

Group mean tail intensity% (SD) #

Group mean of median tail intensity% (SD) #

Vehicle

-

900

2.41 (0.5)

0.34 (0.2)

DAPD

250

900

2.36 (0.3)

0.68 (0.4)

DAPD

500

900

2.43 (0.3)

0.51 (0.1)

DAPD

1000

900

2.37 (0.3)

0.53 (0.2)

EMS

200

450

53.87 (5.3)

54.92 (4.6)***L+

Table 2. Comets - Glandular stomach

Treatment

Dose
(mg/kg/day)

Number of cells scored

Group mean tail intensity% (SD) #

Group mean of median tail intensity% (SD) #

Vehicle

-

900

5.93 (0.9)

2.57 (1.1)

DAPD

250

900

5.71 (0.7)

2.55 (1.3)

DAPD

500

900

11.63 (1.0)

8.87 (1.4)***

DAPD

1000

900

17.91 (1.4)

14.44 (1.9)***

EMS

200

450

55.43 (6.3)

56.84 (7.5)***L+

 

Table 3 - Urinary Bladder

Treatment

Dose
(mg/kg/day)

Number of cells scored

Group mean tail intensity% (SD) #

Group mean of median tail intensity% (SD) #

Vehicle

-

900

1.67 (0.4)

0.37 (0.1)

DAPD

250

900

1.86 (0.2)

0.22 (0.1)

DAPD

500

900

1.71 (0.5)

0.29 (0.2)

DAPD

1000

900

1.61 (0.3)

0.26 (0.2)

EMS

200

450

49.44 (3.4)

50.51 (3.5)***L+

Vehicle- Corn oil

EMS- Ethyl methanesulphonate, positive control (comet phase), dosed once approximately 4 hours prior to termination

SD - Standard Deviation

# Occasional apparent errors of ± 1% may occur due to rounding of values for presentation in the table

Statistical analysis performed on median tail intensity values only

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities): p values for comparisons with control using Williams’ test, unless indicated otherwise (+ t-test)

*** -p< 0.001 (significant)

Otherwise -p> 0.05 (not significant)

L – Analysis performed upon logarithmically transformed data

 

 

Conclusions:
A Comet assay was performed in which rats were dosed with 250, 500 and 1000 mg/kg bw/d of the test item.
It is concluded that DAPD did not show any evidence of causing an increase in DNA strand breaks in the liver and the urinary bladder of male Crl: CD(SD) rats administered DAPD at dose levels up to 1000 mg/kg/day when administered orally by gavage in this in vivo test procedure. In the glandular stomach at the dose level of 1000 mg/kg/day, evidence of causing an increase in DNA strand breaks was noted. However, histopathology findings suggest that the increases in DNA damage observed may be as a result of direct irritation on the glandular stomach. Therefore, the DNA strand breaks observed in the glandular stomach are inconclusive for a true genotoxic response.
Executive summary:

This study was designed to assess the potential of DAPD to induce DNA strand breaks in the liver, glandular stomach and urinary bladder of Crl: CD(SD) rats. Animals were treated with DAPD orally by gavage on two occasions, the second dose being administered approximately 24 hours after the first dose and 3 hours before sampling. All animals were dosed orally by gavage.

The vehicle control and test item dose groups were dosed using a dose volume of 20 mL/kg and the positive control group were dosed using a dose volume of 10 mL/kg. On the basis of results from the preliminary toxicity test, dose levels of 250, 500 and 1000 mg/kg/day were selected for the comet test. No substantial differences in toxicity were observed between the sexes in the preliminary toxicity test, therefore, in line with current guidelines the main test was performed using male animals only. The vehicle control group received corn oil and the positive control group received ethyl Methanesulfonate at 200 mg/kg.

Cell suspensions from the tissues were obtained fromanimals in the vehicle control group and in each of the DAPD treated groups 3 hours after administration of the second dose. Cell suspensions from animals in the positive control group were obtained approximately 4 hours after a single dose. Sections of the stomach from the vehicle control and animals administered DAPD at 250, 500 and 1000 mg/kg/day were processed for histopathological examination and assessed for signs of cytotoxicity, necrosis and apoptosis. Following electrophoresis three slides per animal per tissue were analysed for comets. Slides were visualised by staining with SYBR GOLD® via fluorescence microscopy. 150 morphologically normal cells were analysed for the presence of comets per animal per tissue.

DNA strand breaks were assessed by comparing the mean and median % tail intensities (% TI) from DAPD treated animals compared with vehicle control values. The slides were also examined for any overt toxicity, e.g. an increase in background debris and/or an increase in the incidence of excessively damaged cells (i.e. Hedgehog cells). These cells were excluded from the analysis, along with any cells that had unusual staining artefacts.

There were no statistically significant increases in the median % tail intensity (%TI) observed in the liver or urinary bladder of male Crl:CD(SD) rats when administered DAPD at any dose level compared to vehicle control values. The liver group mean and median %TI of all treatment groups were within the current historical control range. The urinary bladder mean % TI of all treatment groups did not exceed the current historical control range. Statistically significant increases in the median % TI were observed in the glandular stomach of male Crl: CD(SD) rats when administered DAPD at 500 and 1000  mg/kg/day (p<0.001) compared to vehicle control values. The group mean % TI value of animals administered DAPD at 1000 mg/kg/day is outside of the current vehicle historical control range, whereas the value obtained at 500 mg/kg bw/d is within the historical control range.The positive control compound, ethyl methanesulfonate, produced statistically significant increases in the median %TI when compared to vehicle control values (p<0.001, t-test). No hedgehog cells were observed inmale Crl: CD(SD) rats administered DAPD at any dose level, in all tissues analysed.

Microscopic findings related to treatment with DAPD were observed in the glandular stomach of male rats given doses of 500 or 1000 mg/kg/day, consisting of minimal to slight focal/multifocal necrosis or erosion of the mucosa, minimal inflammatory cell infiltration of the mucosa and/or submucosa, and minimal to slight submucosal oedema. Additionally, histopathological findings considered related to treatment were seen in co-processed non-glandular stomach (one animal given 250 mg/kg/day and two animals given 500 mg/kg/day) and pylorus (one animal given 1000 mg/kg/day). The spectrum of histopathological findings in the stomach were considered consistent with direct irritation by the test item DAPD, delivered by oral gavage.

It is concluded that DAPD did not show any evidence of causing an increase in DNA strand breaks in the liver and the urinary bladder of male Crl:CD(SD) rats administered DAPD at dose levels up to 1000 mg/kg/day when administered orally by gavage in this in vivo test procedure. In the glandular stomach at the dose level of 1000 mg/kg/day, evidence of causing an increase in DNA strand breaks was noted. However, histopathology findings suggest that the increases in DNA damage observed may be as a result of direct irritation on the glandular stomach. Therefore, the DNA strand breaks observed in the glandular stomach are inconclusive for a true genotoxic response.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro studies

Gene mutations

A key study on 1,4-benzenediamine, N,N’-mixed Ph and tolyl derivs. for gene mutations (Ames reverse mutation test) was performed according to the GLP guidelines and OECD guideline 471 (Stanikowski, 1993). In this main study 5 strains of Salmonella typhimurium and one strain of Escherichia coli were treated with test substance, using the pre-incubation methodology in the absence and in the presence of Aroclor 1254 induced rat liver S9. The top concentration was 1000 microgram/plate but no bacterial growth was already evidenced at the concentration of 167 µg/plate with all the Salmonella strains in the absence and in the presence of S9. However, with Escherichia coli, 1,4-benzenediamine, N,N’-mixed Ph and tolyl derivs. could be tested up to 1000 µg/plate showing slight to moderate toxicity. Although most of the strains showed no increased levels of mutation in the main experiment, the strains TA1538 and TA98 in the first experiment, and the strain TA1538 in the independent repeat experiment showed a statistically significant dose related increase in the number of revertant colonies with a more than 2-fold increase in at least one dose level in the presence of rat liver S9 only. This means that a metabolite of the test substance exhibited a mutagenic effect on bacteria through frameshift mutations.

Additional bacterial gene mutation studies

Additional Ames reverse mutation tests on 1,4-benzenediamine, N,N’-mixed phenyl and tolyl derivatives are available from the literature.

A study performed by Crebelli et al. addressed the mutagenic effects of test substance on one hand and of tyre-plant workers urinary concentrates on the other. (Crebelli et al., 1984a and Crebelli et al., 1985).

The mutagenicity screening of 1,4-benzenediamine, N,N'-mixed phenyl and tolyl derivatives with different S. typhimurium strains showed that the test substance is weakly active in strain TA98 – a frameshift mutator strain – in the presence of metabolic activation.

The results of the urinary monitoring show that, under these experimental conditions, smoking habits but not occupation in the tyre plant investigated are related to detectable urinary mutagenicity. One possible synergistic effect of occupation with smoking was seen when tyre builders who were smokers were compared with control smokers. The biological significance of the observed synergistic effect is, however, questionable because statistical significance was reached only for a small group of employees.

An Ames reverse mutation test was also performed on rubber chemicals and their nitrosation products (Crebelli, 1984b). This study also showed mutagenic activity with the Salmonella typhimurium strain TA98 in the presence of S9 only while TA100 was negative with and without S9. After incubation of the chemicals with nitrite at low pH, the mixed diaryl-p-phenylenediamines became directly mutagenic in both strains, i.e.mutagenic responses were seen both with and without metabolic activation.

For the three additional Ames reverse mutation tests, which are non-GLP studies, only limited details have been published, and not in sufficient details to make an assessment of the reliability of these studies.

Chromosome aberrations

A non-GLP in vitro chromosome aberrations test was performed according to the EPA OPPTS 870.5375 guideline (SanSebastian, 1994). Chinese hamster ovary cells were exposed to 1,4-benzenediamine, N,N’-mixed Ph and tolyl derivs. for 5 hours at 4 and 25 µg/ml in the absence and in the presence of rat liver S9 respectively, and for 24 and 48 hours up to 4 µg/ml in the absence of S9. Cytotoxicity trials were conducted by exposing cells in culture in a range-finder test up to a concentration level of 375 mg/L which was well above the solubility limit of the test substance. But it is not clear if cytotoxicity was measured in the main study and whether sufficient toxicity levels were reached. An increase in chromosome aberrations was found in the presence of S9-mix at the top concentration of 25 µg/ml at the 5 hour treatment schedule, and at 2 and 4 µg/ml at the 24 hour treatment schedule. But these findings were not confirmed in an additional assay. On basis of the lack of reproducibility, the authors of the study concluded that the test substance is not clastogenic in Chinese hamster ovary cells. In view of the contradictory results, further information was considered useful to fully confirm the substances mutagenic profile. This further information was supplied by means of an in-vivo mutagenicity study (see below).

DNA-damage

A key study to assess DNA-damage is the in vitro Unscheduled DNA-synthesis test (UDS) (Jeffrey, 1998). The in vitro UDS test was conducted according to the GLP guidelines and the OECD guideline 482. Hepatocytes from male Fischer 344 rats were isolated and treated during 20 hours with 1,4-benzenediamine, N,N’-mixed Ph and tolyl derivs. in the presence of 3H-thymidine. The cells were then rinsed, fixed and exposed to autoradiographic emulsion. The slides were developed after 7 days and then stained with H&E. Radiographic grains were counted in 50 nuclei per slide, 3 slides per test substance concentration. Net nuclear grain counts (NNG) were calculated by subtracting the average of 3 cytoplasmic grain counts from the nuclear grain counts. There was no evidence of increased NNG counts indicating that in vitro 1,4-benzenediamine, N,N’-mixed Ph and tolyl derivs. was unable to induce DNA-damage in rat hepatocytes.

In vivo studies

In vivo Micronucleus Test

Because of the contradictory results from the in vitro chromosome aberration study, an in vivo micronucleus test was issued (SanSebastian, 1993). This test has been conducted according to the GLP guidelines and the EPA OPPTS 870.5395 guideline. Groups of 5 female and male CD-1 mice were dosed once intraperitoneally with 1,4-benzenediamine, N,N’-mixed Ph and tolyl derivs. at 250, 1250 and 2500 mg/kg . Two deaths were observed in the 250 mg/kg group and 3 deaths in the 1250 mg/kg group, showing that the maximum tolerated dose had been reached or even exceeded. Bone marrow samples were taken at 24, 48 and 72 hours after dosing. There was no reduction in the proportion of PCE/NCE ratio which indicates that there was no bone marrow toxicity. As bone marrow is a highly perfused organ, one might conclude that in view of intraperitoneal treatment and the high mortality rate, 1,4-benzenediamine, N,N’-mixed Ph and tolyl derivs. entered the blood stream and the bone marrow cells were exposed to the test substance. Micronuclei were scored in 1000 polychromatic erythrocytes per mouse, which is lower than the recommended 2000 PCEs, but no increases in micronucleus frequency were found.

It can be concluded that the absence of chromosome damaging potential noted in the robust in vivo micronucleus test, supports the conclusion in the in vitro chromosome aberration test that 1,4-benzenediamine, N,N’-mixed Ph and tolyl derivs. does not induce chromosomal damage. In view of the findings in the chromosome aberration tests, it can be concluded that the test substance has no chromosome damaging properties.

In vivo Comet Assay

A key in vivo Comet assay was designed to assess the potential of DAPD to induce DNA strand breaks in the liver, glandular stomach and urinary bladder of Crl: CD(SD) rats (Barfield, 2017). Animals were treated with DAPD orally by gavage on two occasions, the second dose being administered approximately 24 hours after the first dose and 3 hours before sampling. All animals were dosed orally by gavage. On the basis of results from the preliminary toxicity test, dose levels of 250, 500 and 1000 mg/kg/day were selected for the comet test.

No substantial differences in toxicity were observed between the sexes in the preliminary toxicity test, therefore, in line with current guidelines the main test was performed using male animals only. The vehicle control group received corn oil and the positive control group received Ethyl MethaneSulfonate at 200 mg/kg. Cell suspensions from the tissues were obtained from animals in the vehicle control group and in each of the DAPD treated groups 3 hours after administration of the second dose. Cell suspensions from animals in the positive control group were obtained approximately 4 hours after a single dose. Sections of the stomach from the vehicle control and animals administered DAPD at 250, 500 and 1000 mg/kg/day were processed for histopathological examination and assessed for signs of cytotoxicity, necrosis and apoptosis.

Following electrophoresis three slides per animal per tissue were analysed for comets. Slides were visualised by staining with SYBR GOLD® via fluorescence microscopy. 150 morphologically normal cells were analysed for the presence of comets per animal per tissue. DNA strand breaks were assessed by comparing the mean and median % tail intensities (% TI) from DAPD treated animals compared with vehicle control values. The slides were also examined for any overt toxicity, e.g. an increase in background debris and/or an increase in the incidence of excessively damaged cells (i.e. Hedgehog cells). These cells were excluded from the analysis, along with any cells that had unusual staining artefacts.

There were no statistically significant increases in the median % tail intensity (%TI) observed in the liver or urinary bladder of male Crl:CD(SD) rats when administered DAPD at any dose level compared to vehicle control values. The liver group mean and median %TI of all treatment groups were within the current historical control range. The urinary bladder mean % TI of all treatment groups did not exceed the current historical control range. Statistically significant increases in the median % TI were observed in the glandular stomach of male Crl: CD(SD) rats when administered DAPD at 500 and 1000  mg/kg/day (p<0.001) compared to vehicle control values. The group mean % TI value of animals administered DAPD at 1000 mg/kg/day is outside of the current vehicle historical control range, whereas the value obtained at 500 mg/kg bw/d is within the historical control range. The positive control compound, ethyl methanesulfonate, produced statistically significant increases in the median %TI when compared to vehicle control values (p<0.001, t-test). No hedgehog cells were observed inmale Crl: CD(SD) rats administered DAPD at any dose level, in all tissues analysed.

Microscopic findings related to treatment with DAPD were observed in the glandular stomach of male rats given doses of 500 or 1000 mg/kg/day, consisting of minimal to slight focal/multifocal necrosis or erosion of the mucosa, minimal inflammatory cell infiltration of the mucosa and/or submucosa, and minimal to slight submucosal oedema. Additionally, histopathological findings considered related to treatment were seen in co-processed non-glandular stomach (one animal given 250 mg/kg/day and two animals given 500 mg/kg/day) and pylorus (one animal given 1000 mg/kg/day). The spectrum of histopathological findings in the stomach were considered consistent with direct irritation by the test item DAPD, delivered by oral gavage.

It is concluded that DAPD did not show any evidence of causing an increase in DNA strand breaks in the liver and the urinary bladder of male Crl:CD(SD) rats administered DAPD at dose levels up to 1000 mg/kg/day when administered orally by gavage in this in vivo test procedure. In the glandular stomach at the dose level of 1000 mg/kg/day, evidence of causing an increase in DNA strand breaks was noted. However, histopathology findings suggest that the increases in DNA damage observed may be as a result of direct irritation on the glandular stomach. Therefore, the DNA strand breaks observed in the glandular stomach are inconclusive for a true genotoxic response.

Justification for classification or non-classification

The mutagenic potential of 1,4-benzenediamine, N,N’-mixed Ph and tolyl derivs. has been studied in three in vitro tests. The substance was positive in one reverse mutation test with bacteria (Ames) (Stanikowski, 1993). The results of two additional Ames reverse mutation tests were published by Crebelli et al. (1984a, 1984b, 1985). The tests were performed on urine concentrates and rubber chemicals and on workers urinary concentrates and raw materials. Positive findings were noted but the authors concluded that the detectable mutagenicity was related to smoking habits and not to occupational exposure to the rubber chemicals.

In an Unscheduled DNA Synthesis (UDS) test (Jeffrey, 1998), 1,4-benzenediamine, N,N’-mixed Ph and tolyl derivs. was not genotoxic; it did not induce DNA damage in rat liver cells.

Also a chromosome aberration test with Chinese hamster ovary cells was conducted (SanSebastian, 1994). An increase in chromosome aberrations was found in some cases, but this result was not confirmed in an additional assay. It was also not clear whether sufficient toxicity levels were reached. The authors concluded that the test substance does not cause breaks in the chromosomes and that therefore 1,4-benzenediamine, N,N’-mixed Ph and tolyl derivs. is not a clastogen. Furthermore, this conclusion is supported by the absence of DNA damage in the in vivo study underneath.

The genotoxicity potential of 1,4-benzenediamine, N,N’-mixed Ph and tolyl derivs. has been studied in two in vivo tests, both conducted according to GLP. In a first assay (SanSebastian, 1993), the result was negative in a bone marrow chromosome aberration test with mice. No increase in micronucleus frequency was found. Based on the high doses administered and on the high mortality rate, it can be concluded that the substance had reached the blood stream and that bone marrow cells were well exposed to the substance. In a second assay (Barfield, 2017), a Comet assay in male Sprague-Dawley rats did not show any evidence of causing an increase in DNA strand breaks in the liver and the urinary bladder up to oral dose levels of 1000 mg/kg/day. In the glandular stomach at the dose level of 1000 mg/kg/day, an increase in DNA strand breaks was noted. However, histopathology findings suggest that the increases in DNA damage observed may be as a result of direct irritation on the glandular stomach. Therefore, the DNA strand breaks observed in the glandular stomach are inconclusive for a true genotoxic response.

In conclusion, 1,4-benzenediamine, N,N’-mixed Ph and tolyl derivs. was not a mutagen in one in vivo test with mice and in two in vitro tests with mammalian cells. There is one positive result with bacteria (Ames) which is not supported by two additional Ames tests. Taking into account the quality of the test design of the chromosome aberration study, the result of this in vivo test overrules the positive result of the in vitro test with bacteria. The in vivo Comet assay was negative for liver and urinary bladder and inconclusive for the glandular stomach. Therefore,1,4-benzenediamine, N,N’-mixed Ph and tolyl derivs. should not be classified for germ cell mutagenicity.